Citation: de Lucas, Á.G.;
Lamminmäki, U.; López-Picón, F.R.
ImmunoPET Directed to the Brain: A
New Tool for Preclinical and Clinical
Neuroscience. Biomolecules 2023, 13,
164. https://doi.org/10.3390/
biom13010164
Academic Editor: Hervé Boutin
Received: 21 December 2022
Revised: 8 January 2023
Accepted: 10 January 2023
Published: 13 January 2023
Copyright: © 2023 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
biomolecules
Review
ImmunoPET Directed to the Brain: A New Tool for Preclinical
and Clinical Neuroscience
Ángel García de Lucas 1,2,3,* , Urpo Lamminmäki 3 and Francisco R. López-Picón 1,2
1 PET Preclinical Imaging Laboratory, Turku PET Centre, University of Turku, 20520 Turku, Finland
2 MediCity Research Laboratory, University of Turku, 20520 Turku, Finland
3 Department of Life Technologies, University of Turku, 20520 Turku, Finland
* Correspondence: angel.garciadelucas@utu.fi
Abstract: Immuno-positron emission tomography (immunoPET) is a non-invasive in vivo imaging
method based on tracking and quantifying radiolabeled monoclonal antibodies (mAbs) and other
related molecules, such as antibody fragments, nanobodies, or affibodies. However, the success
of immunoPET in neuroimaging is limited because intact antibodies cannot penetrate the blood–
brain barrier (BBB). In neuro-oncology, immunoPET has been successfully applied to brain tumors
because of the compromised BBB. Different strategies, such as changes in antibody properties, use of
physiological mechanisms in the BBB, or induced changes to BBB permeability, have been developed
to deliver antibodies to the brain. These approaches have recently started to be applied in preclinical
central nervous system PET studies. Therefore, immunoPET could be a new approach for developing
more specific PET probes directed to different brain targets.
Keywords: positron emission tomography; neuroimaging; immunoPET; brain; blood–brain barrier;
antibody engineering; neuroscience; neuro-oncology; neurology; neuropsychiatry
1. Introduction
Immuno-positron emission tomography (immunoPET) is a non-invasive in vivo imag-
ing method based on tracking and quantifying radiolabeled monoclonal antibodies (mAbs)
and other related molecules, such as antibody fragments, nanobodies, or affibodies [1,2].
Antibody imaging provides a specific and sensitive means of non-invasively characterizing
the cell surface phenotype in vivo, which aids in diagnosis, prognosis, therapy selection,
and monitoring of treatment for many diseases [3]. Since its inception, the field of im-
munoPET has been focused on oncology research because mAbs play an important role in
the clinical management of cancer [4,5]. In addition to cancer, this technology is attractive
for improving our knowledge in the diagnosis of brain disorders, as it is difficult to obtain
biosamples from the brain [6]. However, the success of immunoPET in neuroimaging is
limited because intact antibodies poorly penetrate the blood–brain barrier (BBB). Due to
the poor permeability of the BBB, the delivery of therapeutic bioactive macromolecular
compounds to the central nervous system (CNS) is difficult to achieve [7]. For example,
only 0.1% of peripherally administered antibodies are delivered into the mouse brain [8].
To overcome this problem, different non-invasive methods have been developed to enhance
antibody delivery to the CNS.
PET imaging studies of the CNS initially focused on global or regional changes in
brain function, such as glucose utilization, cerebral blood flow, and oxygen metabolism.
However, according to our knowledge of the diversity of neurotransmitter systems has
been growing, we need to develop molecular imaging probes with an even higher target
specificity than first-generation PET radiotracers [9]. Similar to CNS drug discovery, PET
radiotracer development for the brain is scientifically challenging, with success being
quite sporadic. Primarily, the radiotracer must show high affinity and selectivity for the
target protein. Moreover, the radiotracer must be able to reach its target in vivo [10].
Biomolecules 2023, 13, 164. https://doi.org/10.3390/biom13010164 https://www.mdpi.com/journal/biomolecules
Biomolecules 2023, 13, 164 2 of 16
Therefore, the immunoPET approach could accelerate PET research in the brain to increase
the development of new and more specific imaging probes.
The purpose of this literature review is to outline the major contributions of im-
munoPET to preclinical and clinical neurosciences. First, we write about strategies to cross
the BBB, antibody design, and radiolabel. After that, we summarize the principal advances
in neuro-oncology and neurological diseases. Finally, we hypothesize the development of
immunoPET in these areas and the future possibilities for neuropsychiatry. Other extensive
recent reviews can be found in the diagnosis of glioblastoma diagnosis [11] and Alzheimer’s
disease research [6].
2. Strategies to Cross the BBB
Different strategies have been used to deliver antibodies into the brain, including
modifications in their physicochemical properties; physiological mechanisms, such as
adsorption-mediated transcytosis (AMT) or receptor-mediated transcytosis (RMT); and
induced changes in BBB permeability (Table 1).
Table 1. Summary of different strategies to cross the BBB with proteins or antibodies.
Strategy Mechanism References
Physicochemical properties modification Poly(ethylene glycol) conjugation to increasethe circulation half-life [12,13]
Through physiological mechanism
Cationic proteins or nanobodies that trigger
adsorption-mediated transcytosis (AMT) [14–16]
Ligands or antibodies that trigger
receptor-mediated transcytosis (RMT) [17–32]
BBB permeability changes Open BBB with solvents such as mannitol [19,33]
Focused ultrasound (FUS) with microbubbles [34–38]
One physicochemical modification strategy is to increase the circulation half-life of
antibodies, such as coupling poly(ethylene glycol) (PEG) to antibodies [12]. Prolonged
circulation results in greater serum concentrations, which drive a greater accumulation
of antibodies in low-permeability organs, such as the brain. In addition, PEG can act as a
ligand for cholesterol transport receptors to promote the active transport of macromolecules
into the brain [13].
Various cationic proteins can cross the BBB through AMT [14]. Upon electrostatic
interactions between the cationized protein and anionic charges present on the surface of
epithelial cells of BBB, the endocytosis of cationized proteins is triggered, a preliminary
step for the protein to cross the BBB [15]. The variable heavy-chain domain of camel
homodimeric antibodies (VHH) or nanobodies with a high isoelectric point (pI) have
demonstrated an ability to cross the BBB through this mechanism [15,16].
RMT begins with the binding of a ligand to its cognate receptor on the luminal
membrane of the brain‘s microvascular and capillary endothelial cells. It is a multistage
process involving receptor-mediated endocytosis, mediated by clathrin-coated or non-
clathrin-coated vesicles, followed by intracellular trafficking and vesicular sorting, finally
resulting in vesicle fusion with the abluminal membrane of the BBB and delivery of the
contents to the brain parenchyma [17]. Several receptors capable of inducing RMT are
present in the BBB, such as the insulin receptor, transferrin receptor (TfR), and receptors
responsible for lipoprotein transport, whereas others, such as the albumin receptor, are
not expressed [18]. Antibodies can be modified to pass into the brain by conjugation with
specific BBB receptor ligands [19]. For instance, antibodies targeting the transferrin receptor
have been reported to cross the BBB [20]. Bivalent binding to TfR induces lysosomal sorting
and degradation consistent with the incomplete transcellular trafficking observed in vivo,
but monovalent binding leads to successful transcytosis in the BBB [21]. This brain shuttle
strategy has been used successfully for immunoPET in animal models of Alzheimer’s,
which are reviewed below [22–32].
Biomolecules 2023, 13, 164 3 of 16
The first attempt to modify the permeability of the BBB was in 1981 using mannitol,
which was administered together with a drug, methotrexate, to enhance its delivery to brain
tumors [33]. In the same way, other solvents, such as ethanol, dimethylsulfide, etoposide,
or histamine, have been used to open the BBB and facilitate the delivery of drugs to the
brain. However, the opening of the BBB is nonselective; thus, the use of these agents to
affect the permeability of the BBB can cause serious side effects [19]. A more non-invasive,
local, and targeted way to disrupt the BBB is with focused ultrasound (FUS). This technique,
in combination with microbubbles, can lead to the transient and focal opening of the BBB,
enabling the passage of therapeutic agents across the BBB without relying on the enhanced
permeability and retention effect (EPR) [34]. Antibodies have successfully been delivered
into the brain using FUS [35–38]. In addition, two studies have used an immunoPET
approach to detect radiolabeled antibodies in gliomas [37,38].
3. Antibody Engineering Strategies for ImmunoPET
Antibodies‘ capacity to cross the BBB, which is inherently limited for native antibod-
ies [39], can be significantly improved by endowing the antibody with the capability to
use RMT. This is achieved by generating bispecific antibodies that can bind both the RMT-
associated receptor, such as TfR or insulin receptor and the target of interest in the CNS. The
modular structure of the antibody molecule makes it possible to create bispecific antibodies
of many different designs [40], including asymmetric IgG-like constructs in which the bind-
ing specificity of one of the two inherently identical binding sites has been altered [41–43].
In another common type of construct, antibody fragment(s) providing the second binding
specificity are genetically fused to IgG [24,44,45]. Typically, single chain Fv (scFv) fragments
consisting of the light and heavy chain variable domains linked together with a peptide
linker of approximately 15–20 amino acids are used in such constructs. Yet another way to
generate bispecific constructs is to conjugate two or more antibody fragments with different
binding specificities together with peptide linkers [29,46,47]. Figure 1 shows an example
of each of these three types of the bispecific antibody. Recently, Kariolis et al. reported a
strategy to endow IgG with a capacity to bind TfR for RMT without modifying or adding
variable domains for the second binding specificity. Instead, they engineered the surface of
the C-terminal domain of the Fc-part to introduce a binding site for TfR [48]. In addition to
the use of genetic engineering, chemical conjugation has also been employed for generating
bispecific binders [22].
In addition to IgGs or fragments of them, other types of bioaffinity proteins can be
utilized for the generation of bispecific targeting proteins, including single immunoglobulin
domain-based binders obtained from camelid animals [49] or sharks [31] and alternative
protein scaffolds, such as affibodies [2] and DARPins [50]. For bispecific binding, two or
more such binders can be linked together with peptide linkers, or these binders can be
linked to IgG instead of scFvs. In addition to being smaller (5–20 kDa) than scFvs, the
alternative scaffold-based binders present various other benefits over antibody fragments,
including higher stability and good expression properties. Overall, the ability to use a
variety of antibody formats, alternative binders, and their potential combinations opens up
a plethora of options for developing bispecific bioaffinity molecules with different types of
structural and functional characteristics [51].
The bispecific designs can be assigned to two categories based on the presence or
absence of the Fc-part of immunoglobulin. The presence of the Fc-part increases the circu-
lation half-life of the antibody construct considerably due to FcRn-mediated rescue from
lysosomal degradation. The Fc also contributes to the size of the construct by approxi-
mately 50 kDa. Overall, the Fc-part, whether present or absent, has a significant effect
on the distribution and clearance of antibody constructs in the body [52] and affects the
performance of antibody-based imaging agents.
Another important feature to consider in the bispecific designs is the binding valency
(e.g., the number of binding sites for a specific antigen in the construct). Typically, there
is either one or two binding sites of each specificity in a bispecific construct. Due to the
Biomolecules 2023, 13, 164 4 of 16
bivalency of native IgG, the use of an asymmetric design is typically required to obtain
monovalent binding with an IgG-based construct (Figure 1A). Compared to monovalent
binding, bivalent binding can significantly increase the strength of the interaction with
multimeric targets [53]. On the other hand, even in the presence of more than one binding
site of certain specificity, the dimensions and flexibility of the constructs can affect the
availability of the binding sites within it and, thus, the antibody´s mode of binding. For
example, a construct with two TfR binding scFvs linked to the C-terminus of the light chains
of IgG with short peptide linkers exhibits monovalent interaction with the dimeric TfR,
suggesting that the limited flexibility of the construct hinders the simultaneous binding of
the two scFvs [24].
Biomolecules 2023, 13, x FOR PEER REVIEW 3 of 15 
 
monovalent binding leads to successful transcytosis in the BBB [21]. This brain shuttle 
strategy has been used successfully for immunoPET in animal models of Alzheimer’s, 
which are reviewed below [22–32].  
The first attempt to modify the permeability of the BBB was in 1981 using mannitol, 
which was administered together with a drug, methotrexate, to enhance its delivery to 
brain tumors [33]. In the same way, other solvents, such as ethanol, dimethylsulfide, 
etoposide, or histamine, have been used to open the BBB and facilitate the delivery of 
drugs to the brain. However, the opening of the BBB is nonselective; thus, the use of these 
agents to affect the permeability of the BBB can cause serious side effects [19]. A more non-
invasive, local, and targeted way to disrupt the BBB is with focused ultrasound (FUS). 
This technique, in combination with microbubbles, can lead to the transient and focal 
opening of the BBB, enabling the passage of therapeutic agents across the BBB without 
relying on the enhanced permeability and retention effect (EPR) [34]. Antibodies have 
successfully been delivered into the brain using FUS [35–38]. In addition, two studies have 
used an immunoPET approach to detect radiolabeled antibodies in gliomas [37,38]. 
3. Antibody Engineering Strategies for ImmunoPET  
Antibodies' capacity to cross the BBB, which is inherently limited for native antibod-
ies [39], can be significantly improved by endowing the antibody with the capability to 
use RMT. This is achieved by generating bispecific antibodies that can bind both the RMT-
associated receptor, such as TfR or insulin receptor and the target of interest in the CNS. 
The modular structure of the antibody molecule makes it possible to create bispecific an-
tibodies of many different designs [40], including asymmetric IgG-like constructs in which 
the binding specificity of one of the two inherently identical binding sites has been altered 
[41–43]. In another common type of construct, antibody fragment(s) providing the second 
binding specificity are genetically fused to IgG [24,44,45]. Typically, single chain Fv (scFv) 
fragm ts c nsisting of the light and he vy chain variable domains ink d together with 
a peptide li ker of approximately 15–20 amino a ids are used in such constructs. Yet an-
other way to generate bispecific constructs is to conjugate two or more antibody fragments 
with diff rent binding specificities together with peptide linkers [29,46,47]. Figure 1 shows 
an xample of each of these three types of the bispecif c antibody. Recently, K riolis e  al. 
reported a strategy to endow IgG with a capacity to bind TfR f r RMT with ut modifying 
or dding variable domains for the second binding specificity. Instead, they en ineered 
the surface of t e C-t rminal domain of the Fc-part to i troduce a binding sit  for TfR [48]. 
In addition to the use of genetic engineering, chemical conjugation h s also een em-
ployed for generating bispecific binders [22]. 
 
Figure 1. Examples of bispecific antibody constructs. (A) IgG-like asymmetric antibody engineered 
to bind an RMT-associated target with one of its binding arms and the CNS-associated target with 
the other binding arm. The knob-into-hole approach [41] is used to guide correct pairing of two 
different heavy chains, and CrossMab technology [42] is used to prevent mispairing of the light 
Figure 1. Examples of bispecific antibody constructs. (A) IgG-like asymmetric antibody engineered
to bind an RMT-associated target with one of its binding arms and the CNS-associated target with
the other binding arm. The knob-into-hole approach [41] is used to guide correct pairing of two
different heavy chains, and CrossMab technology [42] is used to prevent mispairing of the light
chains. (B) IgG-like antibody with scFv fragments of different binding specificity conjugated to the
C-terminus of the light chains. This is a symmetric IgG-based construct. (C) di-scFv construct in
which two scFvs with different binding specificities are fused together via a short peptide linker.
VL and VC refer to the light chain variable and constant domains, respectively. VH, CH1, CH2 and
CH3 refer to the heavy chain variable domain and the constant domains 1, 2 and 3, respectively. The
roman numbers in the domain names (e.g., VL-I or VL-II) show which of the two different binding
specificities the domain is associated to.
Complex artificial antibody designs, typical in bispecific antibodies, tend to have an
elevated risk of liabilities related to their biochemical or biophysical properties, including
limited stability, low expression yields, a tendency to aggregate or poor pharmacokinet-
ics [51,54]. These issues can sometimes be tackled or alleviated by further optimizing the
antibody via genetic engineering, but the process can be time-consuming.
Genetic engineering can also be used to modify the binding sites of the antibody to
optimize the strength of the binding. The two different binding sites of a bispecific antibody
can differ in terms of the optimal binding affinity. When it comes to binding the actual
imaging target, high-affinity binding is generally considered to be beneficial for successful
immunoPET imaging. The situation can be different with the recognition of the RMT target;
too high an affinity towards TfR can make the antibody stick to the receptor too tightly [20],
increasing the likelihood of its lysosomal degradation instead of RMT. On the other hand, a
too-weak affinity can impede the accumulation of antibodies in the cells of the BBB.
4. Radiolabeling Strategies
PET scans detect positron-emitting radionuclides, which are attached to a molecule
such as a protein or an antibody. The positron is created, being short-lived, and eventually
gets annihilated, converting all its mass into energy and thereby emitting two photons of
Biomolecules 2023, 13, 164 5 of 16
511 keV each (which is the resting energy of the electron or positron) in opposite directions.
These two photons are detected in coincidence by scintillation detectors of PET scan [55].
Key to the labeling of antibodies, and antibodies-related molecules, is the appropriate
matching between the biological half-life of the protein and the physical half-life of the
radionuclide [2,56]. The shorter-lived PET radionuclides 11C, 18F, 68Ga, 44Sc, and 64Cu
have been used for radiolabeling antibody fragments, while the slow pharmacokinetics
of intact antibodies have enabled the use of the long-lived nuclides 89Zr and 124I [2].
PET radionuclides characteristics used to label antibodies and antibody fragments are
summarized in Table 2.
Table 2. Summary of PET radionuclides characteristics used in immunoPET. Data extracted from [57,58].
Radionuclide Half-Life BranchingRatio (B+) (%)
Positron
Energy–E Max [Mev]
Mean Positron
Range (mm)
11C 20.4 min 99 0.97 1.2
18F 109.7 min 97 0.65 0.6
68Ga 67.7 min 89 1.90 3.5
44Sc 3.97 h 94 1.47 2.3
64Cu 12.7 h 18 0.65 0.7
89Zr 78.4 h 23 0.91 1.3
124I 100.2 h 23 1.54 4.4
The labeling of proteins can be performed by direct labeling, the addition of a pros-
thetic group, or using bifunctional chelators. Direct labeling, which nowadays involves
mostly radioiodination, is the method used to label proteins without using intermediates
such as prosthetic groups or bifunctional chelators. Prosthetic groups are small molecules
able to bind with radionucleides in one site of the structure and simultaneously with a
protein at a second site. An example of this procedure is the modification of the protein
to bear unnatural biorthogonal functional groups such as an azide and then by using
“click” chemistry to achieve the radiolabeled biomolecule. Radiometals (68Ga, 44Sc, 64Cu,
and 89Zr) require bifunctional chelators, which are capable of coordinating a metal ion,
and can be attached to the proteins. There are two main types of bifunctional chelators:
cyclic, such as 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) and 1,4,7-
triazacyclononane-1,4,7-triacetic acid (NOTA); and acyclic multidentate, such as desferriox-
amine B (DFO) and diamine-pentaacetic acid (DTPA) [59]. DOTA and NOTA chelation
methods have been the mainstay for 64Cu labeling of antibody fragments. The DFO ligand
is commonly used to chelate 89Zr due to its high chelation yields, mild reaction condi-
tions, and reasonable stabilities [2]. In addition, several studies have shown that DFO, as
well as NOTA, are suitable chelators for radiolabeling of biomolecules with 68Ga [2,60].
Finally, CHX-A”-DTPA (N-[(R)-2-amino-3-(para-isothiocyanato-phenyl) propyl]-trans-(S,S)-
cyclohexane-1,2-diamine-N,N,N′,N”,N”-pentaacetic acid) has been identified as the most
promising choice for 44Sc, as it permits labeling at room temperature within a reasonable
period of time [61].
The long-lived radionuclides, such as 89Zr and 124I, are compatible with the long
circulation of antibodies or antibody fragments, but they can result in high radiation burden
and low image quality due to the long positron range [62]. To try to overcome this, a pre-
targeting approach allows combining long circulating antibodies or antibody fragments
with short-lived PET radionuclides such as 18F or 11C. In the pre-targeting approach, an
antibody is chemically tagged, injected, and allowed enough time to accumulate in the
target regions, and washout from non-target tissues and blood. Afterward, a radiolabeled
small molecule that can rapidly react with the tag in the antibody is injected [63–65]. The
combined use of the “click” reacting trans-cyclooctene (TCO) derivatives as chemical tags,
Biomolecules 2023, 13, 164 6 of 16
and tetrazine (Tz) derivatives as radiolabeled small molecules, are the state of the art for
pre-targeted PET imaging [66].
Recently developed bi-specific antibodies that penetrate the BBB by targeting trans-
ferrin [22–32] could be labeled with TCO for a pre-targeting approach across the BBB. The
various 18F-labeled Tzs used for pre-targeting approaches in rodent cancer models and
that could be used for neuroimaging studies, unfortunately, do not cross the BBB due
to unfavorable characteristics [67]. Very recently, Shalgunov et al. developed a series of
18F-labelled Tzs that successfully penetrated the BBB in rodents and clicked in vivo with a
TCO-polymer injected into the brain. The lead compound in this study is a promising tool
for future pre-targeted neuroimaging studies [68].
5. Current Applications
5.1. Neuro-Oncology
In 2010, brain metastases were first visualized in HER2-positive breast cancer patients
using [89Zr]Zr-DFO-trastuzumab. This study demonstrated brain lesions with an 18-fold
higher [89Zr]Zr-DFO-trastuzumab uptake in tumors than in normal brain tissue. The brain
penetration of [89Zr]Zr-DFO-trastuzumab was possible because of a disruption of the BBB
at the site of the brain metastasis [69]. Another study in HER2-positive breast cancer
patients using [89Zr]Zr-DFO-pertuzumab demonstrated the detection of brain metastases
in these patients [70]. In HER2-negative metastatic breast cancer patients, immunoPET
following a pre-targeting approach against carcinoembryonic antigen (CEA) showed higher
overall sensitivity than [18F]FDG PET imaging in disclosing metastases, including brain
dissemination [71]. In rodents, HER2-positive intracranial breast carcinoma xenografts
have been shown to uptake of an 18F-labeled single-domain antibody fragment [72].
In addition to brain metastasis, most high-grade gliomas lead to the destruction of
the BBB, with subsequent leakage of contrast medium, which is not shown by low-grade
gliomas [73]. This increased permeabilization in high-grade gliomas, such as glioblastoma
multiforme (GBM), could be used to deliver antibody-based therapeutics or immunoPET
probes. In this sense, several preclinical studies have been conducted with different targets:
integrin αvβ3 [74], epidermal growth factor receptor (EGFR) [61,75,76], delta-like ligand 4
(Dll4) [77], cluster of differentiation 105 (CD105) [75], fibroblast activation protein alpha
(FAP) [78], CD146 [79,80], membrane-type 1 matrix metalloproteinase (MT1-MMP) [81],
CD11b [82], vascular endothelial growth factor (VEGF) [83–85], transforming growth
factor-β (TGF-β) [86,87], and prostate-specific membrane antigen (PSMA) [88,89]. In
the early stages of this field, some studies with different immunoPET approaches were
conducted in GBM flank xenograft models [61,74,75,77–79]. In addition to these models,
orthotopic xenograft mice models were used to demonstrate the capacity of [64Cu]Cu-
NOTA-YY146 to penetrate the disrupted BBB and efficaciously target CD146 within brain
tumors, demonstrating that expression levels of CD146 can be scrutinized non-invasively
in high-grade gliomas with PET imaging for potential patient selection and stratification
for targeted therapies [80]. Another study that used orthotopic GBM xenograft models
demonstrated the feasibility of using an immunoPET probe ([89Zr]Zr-DFO-LEM2/15)
on MT1-MMP marker to visualize GBM tumors for diagnostic purposes. [89Zr]Zr-DFO-
LEM2/15 was able to detect orthotopically growing GBM implants from TS543 (Figure 2A)
but not U251, which correlates with the integrity of the BBB, as analyzed by Evans blue
staining [81]. Therefore, the integrity of the BBB is one of the major limitations for the use
of antibodies in high-grade gliomas because all GBM have clinically significant regions
of tumor with an intact BBB [90]. In addition to targeting tumor cells, tumor-associated
myeloid cells (TAMCs) can be an interesting immunoPET target because, in GBM, the
immunosuppression is largely mediated by these cells. The [89Zr]Zr-DFO-anti-CD11b
antibody PET imaging probe enabled the surveillance of immunosuppressive TAMCs in
the tumor microenvironment of a mouse orthotopic glioma model, thereby providing a
means of assessing the efficacy of immunotherapy [82].
Biomolecules 2023, 13, 164 7 of 16   x FOR PEER REVIEW 9 of 5 
 
 
Figure 2. Examples of immunoPET images in preclinical and clinical neuroscience. (A) Representa-
tive fused PET/CT images of coronal and sagittal planes at 2 and 4 days post-injection containing 
TS543 brain tumors with [89Zr]Zr-DFO-LEM2/15. (Image: Adapted from Ref. [81]). (B) PET/MR fu-
sion images of four different pediatric patients with DIPG where four tumors showed variable up-
take of [89Zr]Zr-DFO-bevacizumab (arrows). (Image: Adapted from Ref. [84]). (C) Representative in 
vivo PET images from TgF344-AD and WT rats 3 days post-administration of [124I]I-OX265-F(ab´)2-
Bapi. (Image: Adapted from Ref. [32]). 
ImmunoPET of neuroinflammation has been studied in the context of Alzheimer’s 
disease. A bispecific antibody based on the triggering receptor expressed on myeloid cells 
2 (TREM2), a marker of microglial activation, and 8D3 scFv against transferrin was radio-
labeled with iodine-124. Radiolabeled mAb1729-scFv8D3CL showed differences in ex 
vivo autoradiography but not in PET. Therefore, other antibody formats or high-affinity 
antibodies must be developed to obtain clear PET imaging [97].  
Multiple sclerosis (MS) is an immune-mediated neurological disorder in which im-
munoPET has been used. Anti-CD20 mAb has shown promising results in patients with 
relapsing-remitting MS [98–100]. However, no clinical tool exists to evaluate whether 
there is a preferential benefit in patients with B-cell disease. Recently, two preclinical re-
search studies aimed to develop antibody-based PET probes against CD20 [101,102]. In 
early research, [64Cu]Cu-DOTA-rituximab showed human B cells in the spinal cord and 
brain of humanized autoimmune encephalomyelitis (EAE) mice model [101]. In the other 
study, 89Zr-labeled-anti-CD20 mAb was injected subcutaneously or intravenously in EAE 
and control mice. Regardless of the route of administration, biodistribution to all major 
organs was consistent in EAE and control mice. However, initial tracer uptake was signif-
icantly higher in the draining lymph nodes and parts of the CNS when administered sub-
cutaneously compared to intravenously in EAE mice [102]. 
6. Perspectives 
The imaging diagnostic tools currently employed in neuro-oncology, computed to-
mography (CT) and magnetic resonance imaging (MRI), provide excellent anatomical in-
formation on the localization of brain lesions but are frequently not able to differentiate 
tumor tissue from other concurrent processes, such as inflammation, edema, or bleeding, 
resulting in under- or over-estimation of the actual extension of the tumor [82]. In addi-
tion, following radiation and/or chemotherapy, neuro-oncologists often encounter treat-
ment-related changes, such as pseudoprogression or necrosis [103]. Another important 
issue in neuro-oncology is that the majority of GBM have gross tumor burden with an 
intact BBB that extends beyond the disrupted BBB. Therefore, successful treatment is only 
possible if an effective drug is delivered with adequate exposure to the entire population 
of targeted cells [91]. The development of new antibody-based PET probes that can cross 
the BBB could improve the diagnosis of brain tumors and also be a great tool for managing 
immune and radiation therapy.  
Figure 2. Examples of immunoPET images in preclinical and clinical neuroscience. (A) Representative
fused PET/CT images of coronal and sagittal planes at 2 and 4 days post-injection containing TS543
brain tumors with [89Zr]Zr-DFO LEM2/15. (Image: Adapted from Ref. [81]). (B PET/MR fusion
images of four different p diatric patients with DIPG where four tumors sh wed variable uptake of
[89Zr]Zr-DFO-bevacizumab (arrows). (Image: Adapte from Ref. [84]). (C) Representative in vivo
PET images from TgF344-AD and WT rats 3 days post-administration of [124I]I-OX265-F(ab´)2-Bapi.
(Image: Adapted from Ref. [32]).
One interesting example of fast translation from the preclinical to the clinical field is
[89Zr]Zr-DFO-bevacizumab PET imagi g in diffuse intrinsic pontine glioma (DIPG) [83–85].
Bevacizumab is a rec mbinant humanized monoclonal antibody ag inst VEGF that has
demonstrated sig ificant resp nses and prolonged survival in individual patients with
DIPG [91,92]. Therefore, the challenge is to identify patients who will benefit from treat-
ment w th bevacizumab [84]. In the first r search paper, [89Zr]Zr-DFO-bevacizumab was
used to s udy its bi distribut on in different intracranial and subcutaneous murine tumor
odels, and accum lation of this antibody was nly observed in the subcutaneous tu-
mor models. These results are in line ith the poor clinical response rates obtained with
bevac zumab thus far in children with DIPG. Consequently, this research suggests that
bevacizumab treatment is only justified if the tumor has be n previously detected using
[89Zr]Zr-DFO-bevacizumab [83]. One ye r later, a first pilot immunoPET study in pe iatric
DIPG patients suggested that the addition of [89Zr]Zr-DFO-b vacizumab PET imaging
(Figure 2B) may help in selecting potential candidates for bevacizumab treatment of DIPG
because this procedure assesses both target availability and drug accessibility of the tu-
mor [84]. Subseque tly, a study was performed with a 1-on-1 analysis of multiregio al
in vivo and ex vivo [89Zr]Zr-DFO-bevacizumab uptake, tumor histology, and vascular
morphology in a DIPG patient. PET imaging was capable of detecting heterogeneity in
[89Zr]Zr-DFO-bevacizumab uptake between lesions, which correlated well with the ex vivo
measurements. However, PET cannot detect subcentimeter intralesional uptake hetero-
geneity [85]. Another clinical study demonstrated that a radiolabeled antibody against
TGF-β, [89Zr]Zr-DFO-fresolimumab, reaches recurrent high-grade gliomas. However,
monotherapy with fresolimumab did not result in an antitumor effect [86]. Interestingly, a
recent preclinical research paper in which [89Zr]Zr-DFO-fresolimumab was used to detect
radiation-induced TGF-β activation in tumors suggested that fresolimumab could improve
the outcome of radiotherapy [87]. Finally, anti-PSMA radiolabeled antibody, [89Zr]Zr-
DFO-huJ591, was studied in a 51-year-old woman diagnosed 8.5 years ago with grade
II oligodendroglioma (1p/19q co-deleted, IDH mutant) and disease progression despite
multiple prior treatments, including surgery and chemo-radiation, most recently with IDH
inhibitor AG-120 (NCT02074839). [89Zr]Zr-DFO-huJ591 uptake was observed in two brain
lesions determined to be anaplastic oligodendroglioma (grade III) histologically [88]. In
addition to intact antibodies in clinical research, a radiolabeled minibody against PSMA,
[89Zr]Zr-DFO-IAB2M, was tested in two high-grade gliomas and metastatic brain tumors
from lung cancer patients. [89Zr]Zr-DFO-IAB2M may have potential value in the differen-
Biomolecules 2023, 13, 164 8 of 16
tial diagnosis of high-grade glioma from primary CNS lymphomas (PCNSL) or radiation
necrosis, as well as in the prediction of treatment efficacy and assessment of the treatment
response to bevacizumab therapy for high-grade glioma [89].
5.2. Neurological Diseases
The first attempts to reach the brain with a preclinical immunoPET approach used
poly(ethylene glycol) antibodies against amyloid-β (Aβ). 64Cu-labeled anti-Aβ mAbs 6E10,
M116, and M31, showed differences in uptake between the TgCRND8 mouse model of
Alzheimer’s disease and wild-type animals [12,13]. In addition, radiolabeled antibodies
without specific modification to cross the BBB were studied in Alzheimer‘s rodent models.
However, limited penetration made them inadequate for monitoring cerebral amyloid
pathogenesis [93,94].
The first successful bispecific antibody-based brain PET study was performed in
2016. A human version of mAb158 F(ab’)2 fragment against soluble Aβ protofibrils was
chemically conjugated to 8D3 TfR antibody and radiolabeled with iodine-124. [124I]I-8D3-
F(ab´)2-h158 was taken up into the brain through TfR-mediated transcytosis, and Aβ was
detected in the tg-ArcSwe and tg-Swe Alzheimer mouse models [22]. Age or genotype did
not influence the systemic pharmacokinetics or biodistribution to major organs [23]. In these
studies, PET imaging correlated with age and Aβ pathology in the brains of Alzheimer‘s
mouse models [22,23]. However, the chemical conjugation resulted in a heterogeneous
mixture of fusion proteins randomly linked together at different positions, which is not
suitable for clinical application. In addition, the use of the complete 8D3 antibody resulted
in bivalent TfR binding, which has been shown to be suboptimal for transcytosis. Therefore,
a new format was created in which the C-terminal end of the RmAb158 light chain was
attached to the scFv of 8D3. In this way, monovalent interactions were achieved with TfR
despite having two binding sites, improving transfer across the BBB, with higher or equal
brain uptake compared to previously reported BBB shuttles at both trace and therapeutic
doses [24]. [124I]I-RmAb158-scFv8D3 demonstrated an ability to follow disease progression
and detect the effects of β-secretase inhibitor treatment by PET imaging in different mouse
models of Alzheimer’s disease and was able to detect quantitative differences between
treated and untreated groups, whereas [11C]Pittsburgh compound B ([11C]PiB) PET did not
detect any differences between treated and untreated groups [25,26]. RmAb158-scFv8D3
and 8D3-F(ab’)2-h158 are large proteins (203 kDa and 270 kDa, respectively) with intact Fc
domains and have long biological half-lives in blood, which make them not well suited
for clinical use as radioligands [29]. For example, RmAb158-scFv8D3 was radiolabeled
with fluorine-18, and PET imaging was performed 12 h after injection, but it was not
enough to detect differences between transgenic mice with Aβ deposits and wild-type
mice [27]. For this reason, smaller recombinant formats without the Fc domain, such as
TribodyTM (100 kDa) or di-scFv (58 kDa), have been created [28–30]. TribodyTM radioligand
is composed of two fragments of mAb158 fused to Fab 8D3. It has a shorter half-life in
blood than RmAb158-scFv8D3 and 8D3-F(ab’)2-h158 (9 h vs. 12 h and 19 h, respectively)
and shown a clear distinction between wild-type and Alzheimer mouse models [27,28].
[124I]I-Di-scFv3D6-8D3 was designed with an scFv derived from an anti-Aβ murine 3D6
antibody. It is capable of crossing the BBB and detecting in vivo intrabrain Aβ with better
sensitivity than [11C]PiB [29]. [125I]I-Di-scFv3D6-8D3 has been measured at similar brain
parenchymal concentrations as [125I]I-mAb3D6-scFv8D3, but with less retention in the
capillaries, probably due to the lower avidity of [125I]I-di-scFv3D6-8D3 against TfR. The
elimination rate from the brain is similar for both [30]. Interestingly, a single domain shark
antibody VNAR fragment (TXB2) with similar affinity to murine and human TfR1 was
studied in PET because 3D8 is an antibody against murine TfR. It was fused to the Aβ
antibody bapineuzumab (Bapi) and showed markedly increased brain uptake compared
to Bapi alone [31]. Furthermore, a recent study with variants of the rat TfR antibody,
OX26, chemically conjugated to F(ab´)2 fragments of Bapi has demonstrated that the
TfR-mediated transport of an immunoPET radioligand enables sensitive imaging of brain
Biomolecules 2023, 13, 164 9 of 16
Aβ pathology in a rat model of AD (Figure 2C) [32].In addition to beta-amyloidopathy,
another proteinopathy, α-synuclein (αSYN) deposition, has been studied by immunoPET.
Several radiolabeled bispecific antibodies were positively studied in an αSYN deposition
model with PET, and the in vitro characterization showed that the antibodies had high
sensitivity towards aggregated αSYN. However, the lack of signal in the transgenic models
(L61 and A30P) could be due to the intracellular localization of the target [95]. Finally, an
apolipoprotein E (ApoE)-derived brain shuttle peptide fused to an 18F-labeled affibody
against monomeric and oligomeric states of αSYN was successfully synthesized. The
results of this study demonstrated that this strategy can potentially be utilized for brain
molecular imaging [96].
ImmunoPET of neuroinflammation has been studied in the context of Alzheimer’s
disease. A bispecific antibody based on the triggering receptor expressed on myeloid
cells 2 (TREM2), a marker of microglial activation, and 8D3 scFv against transferrin was
radiolabeled with iodine-124. Radiolabeled mAb1729-scFv8D3CL showed differences in ex
vivo autoradiography but not in PET. Therefore, other antibody formats or high-affinity
antibodies must be developed to obtain clear PET imaging [97].
Multiple sclerosis (MS) is an immune-mediated neurological disorder in which im-
munoPET has been used. Anti-CD20 mAb has shown promising results in patients with
relapsing-remitting MS [98–100]. However, no clinical tool exists to evaluate whether there
is a preferential benefit in patients with B-cell disease. Recently, two preclinical research
studies aimed to develop antibody-based PET probes against CD20 [101,102]. In early
research, [64Cu]Cu-DOTA-rituximab showed human B cells in the spinal cord and brain of
humanized autoimmune encephalomyelitis (EAE) mice model [101]. In the other study,
89Zr-labeled-anti-CD20 mAb was injected subcutaneously or intravenously in EAE and con-
trol mice. Regardless of the route of administration, biodistribution to all major organs was
consistent in EAE and control mice. However, initial tracer uptake was significantly higher
in the draining lymph nodes and parts of the CNS when administered subcutaneously
compared to intravenously in EAE mice [102].
6. Perspectives
The imaging diagnostic tools currently employed in neuro-oncology, computed to-
mography (CT) and magnetic resonance imaging (MRI), provide excellent anatomical
information on the localization of brain lesions but are frequently not able to differentiate
tumor tissue from other concurrent processes, such as inflammation, edema, or bleeding,
resulting in under- or over-estimation of the actual extension of the tumor [81]. In addition,
following radiation and/or chemotherapy, neuro-oncologists often encounter treatment-
related changes, such as pseudoprogression or necrosis [103]. Another important issue in
neuro-oncology is that the majority of GBM have gross tumor burden with an intact BBB
that extends beyond the disrupted BBB. Therefore, successful treatment is only possible if
an effective drug is delivered with adequate exposure to the entire population of targeted
cells [90]. The development of new antibody-based PET probes that can cross the BBB
could improve the diagnosis of brain tumors and also be a great tool for managing immune
and radiation therapy.
Brain PET imaging allows a varied approach by assessing several physiopathological
pathways, including neuroinflammation, neurotransmission, and protein aggregation, in
neurological diseases [104]. Translocator protein-18 kDa (TSPO) is an 18 kDa outer mito-
chondrial membrane protein that is upregulated in neuroinflammation and a gold standard
for PET imaging of activated microglia [105,106]. However, TSPO is expressed in reactive
astrocytes, and many TSPO tracers have low sensitivity to the A147T variant. In addition,
available TSPO ligands do not distinguish between pro- and anti-inflammatory microglia,
which may be differentially expressed at the onset and progression of a neuroinflammatory
condition [107,108]. Therefore, immunoPET could help develop new PET tracers targeting
other neuroinflammatory biomarkers, such as a cannabinoid-2 receptor, purinergic recep-
tors, or triggering receptors expressed on myeloid cells, among others [107]. Regarding
Biomolecules 2023, 13, 164 10 of 16
neurotransmission, immunoPET could allow the development of specific PET tracers for
targets that have proven to be difficult using small molecules. For example, the possibility
of obtaining in vivo information about the integrity of the cholinergic transmission by PET
imaging is of major interest in studying the pathophysiology of various neurodegenerative
disorders, such as Alzheimer’s disease and Lewy body dementia, and has major relevance
in cholinergic drug development [109]. Nevertheless, one of its key elements, muscarinic
acetylcholine receptors (mAChRs), does not have a PET tracer with clinical applications,
which demands mAChR tracers with improved imaging properties. In addition, M3 and
M5 mAChR subtypes do not have any tracer in an early stage of development [110]. Finally,
Parkinson’s disease, a protein aggregation disease, remains a challenge because clinical
features of the disease overlap with other neurodegenerative conditions, and diagnostic
tests or biomarkers still do not allow for a definitive diagnosis from the earliest stages.
In addition, there is a need to better define Parkinson’s disease subtypes, which not only
have different clinical presentations and prognoses but also differ in the underlying dis-
ease mechanisms, calling for personalized treatment approaches [111]. In this context,
immunoPET could help create highly specific imaging probes against biomarkers, such as
αSYN and TAR DNA-binding protein 43 (TDP-43), for which there are currently no other
clinical PET probes available. It could improve early diagnosis and personalized medicine
in neurodegenerative disorders.
Another field in which immunoPET could bring new perspectives is neuropsychiatry.
No specific marker of a psychiatric disorder has been established thus far [112], and the
development of antibody-based PET probes could help find them. For example, post-
mortem investigations of GABA-A receptors in schizophrenia have shown that the α1
subunit increases and the α2 subunit decrease their expression, whereas the α5 subunit has
inconsistent results. However, the density of GABA-A receptors in schizophrenia does not
appear to be abnormal in radioligand studies, as postmortem studies have demonstrated.
Some authors and we believe that these negative findings can be explained by the lack of
imaging tracers with high affinity and specificity for different subunits [113]. Therefore, the
high specificity of antibodies could be used to detect each of the 19 subunits of GABA-A re-
ceptors. In this way, new immunoPET tools could be developed to look for new biomarkers
in neuropsychiatric disorders.
Recently developed PET technologies, such as multiplexed PET (mPET), long axial
field of view (LAFOV) PET/CT scanners, and hybrid PET/MR imaging, together with
immunoPET probes, could lead to novel knowledge in CNS research and contribute to drug
development. The use of mPET, which can detect two radioisotopes at the same time [114],
could allow the differentiation of, for example, specific GABA-A receptor configurations,
such as α1β2 or α1β3 subunits, at once. This information could give a better picture of
the GABA-A receptor subtype expression in vivo in psychiatric disorders. LAFOV PET
scanners have an extended axial FOV (> 1 m), enabling whole-body dynamic PET imaging,
and have several benefits, such as increased sensitivity, whole-body kinetic analysis, and
lower radiotracer dosing [115,116]. These devices allow physiological or pathophysiological
brain–heart–kidney–liver–gut interactions to be studied [115]. Finally, hybrid PET/MR
allows temporal and spatial matching datasets displaying different physiological and
metabolic information about a disease process. This multimodal technology could provide
an efficient means for acquiring images in neuro-oncology, neurodegenerative disease,
epilepsy, cerebrovascular disease, psychiatry, and neurology and may show added benefit
beyond separate acquisition and interpretation of PET and MRI alone [117].
Current advances in antibody engineering have achieved successful application of
immunoPET to the brain. This achievement opens the doors to improving diagnosis and
treatment in neuro-oncology, neurology, and neuropsychiatry.
Author Contributions: All authors contributed to the original draft preparation and reviewed and
edited the manuscript. All authors have read and agreed to the published version of the manuscript.
Biomolecules 2023, 13, 164 11 of 16
Funding: This paper was supported by the European Union’s Horizon 2020 research and innovation
program under the Marie Sklodowska-Curie (grant 891455) and the Turku University Hospital
(grant 11135).
Conflicts of Interest: The authors declare that the research was conducted in the absence of any
commercial or financial relationships that could be construed as a potential conflict of interest.
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