State of the Art in Radiolabeling of Antibodies with Common and Uncommon Radiometals for Preclinical and Clinical Immuno-PET.

Inert and stable radiolabeling of monoclonal antibodies (mAb), antibody fragments, or antibody mimetics with radiometals is a prerequisite for immuno-PET. While radiolabeling is preferably fast, mild, efficient, and reproducible, especially when applied for human use in a current Good Manufacturing Practice compliant way, it is crucial that the obtained radioimmunoconjugate is stable and shows preserved immunoreactivity and in vivo behavior. Radiometals and chelators have extensively been evaluated to come to the most ideal radiometal-chelator pair for each type of antibody derivative. Although PET imaging of antibodies is a relatively recent tool, applications with 89Zr, 64Cu, and 68Ga have greatly increased in recent years, especially in the clinical setting, while other less common radionuclides such as 52Mn, 86Y, 66Ga, and 44Sc, but also 18F as in [18F]AlF are emerging promising candidates for the radiolabeling of antibodies. This review presents a state of the art overview of the practical aspects of radiolabeling of antibodies, ranging from fast kinetic affibodies and nanobodies to slow kinetic intact mAbs. Herein, we focus on the most common approach which consists of first modification of the antibody with a chelator, and after eventual storage of the premodified molecule, radiolabeling as a second step. Other approaches are possible but have been excluded from this review. The review includes recent and representative examples from the literature highlighting which radiometal-chelator-antibody combinations are the most successful for in vivo application.

Antibodies and Immuno-PET

The Emerging Role of Antibodies

Monoclonal antibodies (mAbs) have emerged as next-generation therapeutic drugs, especially for the treatment of cancer, due to their high specificity toward certain antigens. Ideally, the target should be tumor selective to avoid binding of mAbs to healthy organs expressing the same target.[1] While initially, only unconjugated IgGs (mostly of IgG1 subclass) were developed for therapy, mainly used for blocking signal transduction of tyrosine kinases or other membrane receptor targets involved in oncology and other diseases,[2] the trend in the past decade is set on development of antibody–drug conjugates (ADC), multispecific mAbs, immune checkpoints inhibitors, and also mAb fragments such as single domain antibodies, nanobodies, and antibody mimetics such as affibodies (Figure ; in this review collectively called “antibodies”).[3−5] The development of this wide range of antibodies is accompanied by questions concerning their in vivo behavior, pharmacokinetics, and targeting efficiency. The growing field of mAb development is comprehensively described in the journal mAbs that publishes yearly an update on “antibodies to watch”, showing that between 2010 and the end of 2019, the cumulative number of antibodies approved in the US and EU has almost tripled resulting in a total of about 80 approvals. About 50% of the antibody therapeutics currently in phase II or III clinical studies are evaluated in noncancer applications.[6,7]

Figure 1

Representation of a monoclonal antibody, isotype IgG, containing two light (L) and heavy (H) chains maintained together via disulfide bonds. The variable region (Fv) is composed of the variable heavy (VH) and variable light (VL) chains. VH and VL are together with the constant light and heavy chain 1 (CL and CH1) constituting the Fab region. CH2 and CH3 are the constant region (Fc). Many of the smaller molecular weight antibody fragments have been engineered from this general structure and the ones discussed in this review are summarized here, including the antibody mimetic affibody. Approximative molecular weight and in vivo biological half-life are indicated.

Positron Emission Tomography (PET) as a Tool to Evaluate Antibodies

Positron emission tomography (PET) is not only used for diagnosis and response monitoring using [18F]FDG, but has also become a highly valuable imaging technique helping to understand the behavior of antibodies at an early stage during development by evaluating the radiolabeled antibodies in so-called immuno-PET imaging studies. Immuno-PET is now increasingly used in early-phase clinical trials for making go/no go decisions based on results obtained with a limited number of patients. In this way, immuno-PET enables steering drug design and contributes to drug and patient selection, and as a consequence, the development of new medicines is sped up in a cost-effective way. Thanks to its high resolution, sensitivity, and better quantification ability, PET is preferred over single-photon emission computed tomography (SPECT), while the availability of preclinical and clinical PET cameras strongly increased during the past decade.[8]

Radionuclides for PET Imaging of Antibodies

Antibodies are proteinaceous molecules typically ranging from a few to about 150 kDa in size. As a result, their pharmacokinetics also range from short (less than an hour serum half-life) to long (days) blood circulating half-life. To enable in vivo characterization of an antibody with a PET camera, a positron emitter has to be attached in a stable and inert way. For this purpose, radiometals are particularly interesting, since they can be used in combination with antibodies that have been premodified by means of a bifunctional chelating agent, allowing facile single-step radiolabeling. In addition, radiometals of such constructs possess residualizing properties. Zirconium-89, copper-64, and gallium-68 have emerged over the years as the preferred radiometals for radiolabeling of antibodies, because of their physical properties, availability, costs, and ease of radiolabeling (see Tables and 2).[9,10] The half-life of zirconium-89 (t1/2 = 78.4 h) matches the biological half-life of long-circulating large (slow kinetic) molecules like intact mAbs (Figure ), while copper-64 possesses an intermediate half-life (t1/2 = 12.7 h) that can be used for antibody fragments with medium to relatively fast kinetics. On the contrary, radionuclides such as gallium-68 (t1/2 = 67.7 min) are preferred for antibody fragments and mimetics with a very short serum half-life such as nanobodies or affibodies (see Table ). Although 89Zr, 64Cu, and 68Ga are the most commonly used radiometals for radiolabeling of antibodies, none of them are perfect, triggering research on other emerging radiometals such as manganese-52, yttrium-86, gallium-66, and scandium-44. Moreover, recent advances in [18F]AlF radiochemistry expand the options for radiolabeling of antibodies with 18F. 18F is by far the most commonly used PET radionuclide thanks to its short half-life (110 min), its ideal physical properties as extensively shown in small molecule imaging, and its increasing availability. Finally, the application of matching theranostic pairs (SPECT or PET for imaging combined with radionuclide therapy) is an interesting re-emerging field in nuclear medicine. Around 20 years ago, a first wave has been observed in the use of theranostics, which was followed by near-abandonment.[11] In recent years, there has been renewed interest in theranostics as exemplified by, e.g., PSMA imaging and therapy.[12] The concept of matching pairs relies on two different approaches, either using the same targeting ligand in combination with two different isotopes (a diagnostic and a therapeutic one) of the same element or using two different radionuclides.[13] The first approach is in theory ideal to assess biodistribution, target accumulation, and redistribution of the radioactive and nonradioactive catabolites to fully predict therapeutic response by dosimetric analysis. In the second approach, the diagnostic radiotracer is used as a scouting agent for the therapeutic one, but this requires that both tracers present the same in vivo behavior.

Table 1

Physical Characteristics and Production Route of PET Radionuclides Discussed in This Review

radionuclide	production route	half-life	Eβ max (kev)	mean range in water (mm)	β+ (%)	characteristic main transition γ (keV)
52Mn	Cyclotron 52Cr(p,n) 52Mn	5.6 d	β+ (573.3)	0.6	ε+β+ (100%)	434.1 (100.0%)b
					β+ (29.4%)	935.5 (94.9%)
						744.2 (90.3%)
						333.6 (5.1%)
89Zr	Cyclotron 89Y(p,n) 89Zr	78.4 h	β+ (902)	1.2	ε+β+ (100%)	909 (99.9%)
					β+ (22.7%)
86Y	Cyclotron 86Sr(p,n) 86Y	14.74 h	β+ (3153)	1.9	ε+β+ (100%)	1076.6 (83.0%)b
					β+ (31.9%)	627.7 (32.6%)
						1153.0 (30.6%)
						777.4 (22.4%)
						1920.7 (20.8%)
						1854.4 (17.2%)
						443.1 (16.9%)
						703.3 (15.4%)
						645.9 (9.2%)
64Cu	Cyclotron 64Ni(p,n) 64Cu	12.7 h	β+ (653.0)	0.7	ε+β+ (61.5%)	n.a.a
			β– (579.4)		β+ (17.6%)
					β– (38.5%)
66Ga	Cyclotron 66Zn(p,n)66Ga	9.49 h	β+ (4153)	9.3	ε+β+ (100%)	1039.2 (37.0%)b
					β+ (56.5%)	2751.9 (23.3%)
						833.5 (5.9%)
						2189.6 (5.6%)
44Sc	Cyclotron 44Ca(p,n) 44Sc-generator 44Sc(p,2n) 44Ti → 44Sc	4.0 h	β+(1473.5)	2.3	ε+β+ (100%)	1157.0 (99.9%)
					β+ (94.3%)
18F–Al	Cyclotron 18O(p,n) 18F	109.8 min (18F)	β+ (633.5)	0.6	β+ (96.9%)	n.a.a
68Ga	Generator 68Ge → 68Ga	67.7 min	β+ (1899.1)	2.9	ε+β+ (100%)	n.a.a
					β+ 90%

Nonapplicable: ≤1%

Energies with an abundance <5% left out.

Table 2

Representative Radiolabeling Conditions for Antibodies or Antibody Derivatives Evaluated in Recent Preclinical Studies with 89Zr, 64Cu, 68Ga, and [18F]AlFa

radionuclide	chelator (-linker)	antibody derivatives	conjugation conditions	radiolabeling conditions	ref
89Zr	DFO(-NCS)	mAb	3 equiv; 30 min; 37 °C; pH ∼ 9	1 h; RT; pH ∼ 7	(14,23−27,63)
	DFO(-maleimide)	mAb	60 equiv; 60 min; RT; pH not indicated	1 h; RT; pH ∼ 7	(64)
		Affibodies	34–40 equiv; 2 h; 40 °C; pH ∼ 7.4 (in PBS)	1 h; RT; pH ∼ 7	(65)
	DFO(-N-suc-TFP ester)	mAb	2 equiv; 30 min; RT; pH ∼ 9; Fe removal with EDTA; 30 min; 35 °C pH 4.3–4.5	1 h; RT; pH ∼ 7	(63,66,67)
	DFO*(-NCS)	mAb	3–5 equiv; 30–45 min; 37 °C; pH ∼ 9	1 h; RT; pH ∼ 7	(47,50)
	DFOSq	mAb	3–20 equiv; overnight; RT; pH ∼ 9	25 min–1 h; RT; pH ∼ 7	(50,52,68)
	DFO*Sq	mAb	5 equiv; overnight; RT; pH ∼ 9	1 h; RT; pH ∼ 7	(50)
	3,4,3-(LI-1,2-HOPO)-(NCS)	mAb	5 equiv; 1 h; 37 °C; pH ∼ 9	RT; 1–3 h; pH ∼ 7	(37)
	DFO-cyclo*-(NCS)	mAb	30 equiv; overnight; 37 °C; pH 8.4	30 min; RT; pH ∼ 7	(48)
64Cu	DOTA(-maleimido-monoamide)	Affibodies	15 equiv; 2 h; RT; pH 7.4	1 h; 40 °C; pH 6	(69)
	DOTA(-NCS)	mAb	10 equiv; overnight; 37 °C; pH 8.5	1 h; 40 °C; pH 5.5	(70)
	DOTA(−NHS)	mAb	10–30 equiv; 1 h (or overnight at 4 °C); RT to 37 °C; pH 7.0–8.5	30 min–1 h; 37–43 °C; pH 5.0–7.0	(71−73)
	NOTA-(NCS)	Fab and F(ab′)2	5–10 equiv; 1–2 h, RT to 37 °C; rarely 24 h or overnight at 4 °C; pH 8.0–9.2	30 min (15 min to 1 h); 37–40 °C; mostly pH 5.0 (4.5–5.5)	(74−99)
		scFv	25 equiv; overnight; 4 °C; pH 9	1 h; 40 °C; pH 6.5
		Nanobody	20 equiv; 2 h; RT; pH 8.7	10 min; RT; pH 5
		mAb	Mostly 5–25 equiv; 1–3 h ± overnight at 4 °C; RT to 37 °C; pH 8.0–9.0,	30 min–1 h; RT to 37 °C; pH 4.5–6.5
	NOTA(-NHS)	mAb	50–55 eq ; 1 h RT or overnight at 4 °C; pH 7.5–8.6	1 h; 37–42 °C; pH 5.0.-6.0
	NOTA-maleimide	Diabody	Site specific with maleimide, pH 7.4	1 h; 40 °C; 1 h; pH 6
	NODAGA(-NCS)	Fab	equiv not indicated; 1 h; 37 °C; pH 8.5	20 min; 37 °C; pH 5.5	(100−105)
		F(ab′)2	20 equiv; 3 h; RT; pH 9.5	30 min; RT; pH 5.5
		mAb	20 equiv; 1 h; RT; pH 9	1 h; 52 °C; pH 5–6
		mAb	25 equiv; 16 h; 4 °C; in PBS ∼ pH 7.4	30 min; 42 °C; pH 6–7
	NODAGA(-NHS)	mAb	55 equiv; overnight; 4 °C pH ∼ 7	1 h; 42 °C, pH 7
	PCTA(-NCS)	mAb	10 equiv; RT 2 h then 4 °C overnight; pH 8.5	1 h; RT; pH 6.5	(106−108)
		mAb	5 equiv; overnight; 37 °C; pH 8.5	1 h; 40 °C; pH 5.5
	CB-TE2A with a Gly-Glu-Glu-Glu spacer	Affibodies	10 equiv; 2 h; RT; no pH indicated	45 min; 95 °C; pH 5.6	(109)
	phosphinate PS(-NCS)	mAb	40 equiv; 2 h RT, then 4 °C for 12 h; pH 8.5	40 min; 37 °C; pH 5.5	(110)
	Sarcophagine derivatives	mAb fragments	250 equiv chelator; 500 equiv EDC; RT? (not indicated); 30 min; pH 5	30 min; 25 °C; pH 5	(111)
68Ga	DOTA(-MMADOTA) (maleimide-monoamide)	Affibody	Site-specific, overnight; 37 °C; pH 5.5	15 min, 80 °C; pH 3.9	(112)
	NOTA-(NCS)	mAb	25 equiv; 16 h; RT; pH 9	pH 5.0, 5 min, RT	(113)
		Nanobody	10–20 equiv; 2–2.5 h; RT; pH 8.5–8.7	5–10 min; RT; pH 4.7–5.0,	(114−116)
		F(ab′)2	200 equiv; overnight; 4 °C pH 9	10–15 min; RT to 39 °C; pH ∼ 5.0–5.5	(117,118)
		Single domain antibody	∼10–20 equiv; 2–18 h; RT; pH 8.5–8.7	10 min; RT; pH 4.0–5.0	(119−121)
	NOTA(−NHS)	mAb	3 equiv; overnight; 4 °C; in water (pH ∼ 7)	30 min; RT; pH 3.7	(122)
		scFv	∼3.5 equiv, overnight, 4 °C; pH not indicated	30 min; RT; pH 3.7	(123)
	photoactivable chelate, HBED-CC-PEG3-ArN3	mAb	5 equiv; LED irradiated 10 min, RT; pH 8–9	RT; reaction followed by TLC; pH 4.4	(124)
	DFO comparison with 89Zr DFO-derivative	scFv	3 equiv; 30 min, 37 °C in 50 mM NaHCO3	5 min; RT but low chelator:protein ratio obtained (0.14 DFO-NCS per scFv); pH 5.5	(125)
[18F]AlF	NOTA(-MMA)	Affibody	20 equiv; site specific, 1–2 h, 37 °C; pH 7.4	15 min; 100 °C; pH 4	(126, 127)
	phenyloxadiazolyl methylsulfone derivatives (PODS) chelators: NOTA- and NODAGA-PODS-	Affibody	15 equiv; site specific with maleimide, 1 h; 37 °C; pH 7	15 min; 100 °C; pH 4	(128)
	RESCA as (±)-H3RESCA-TFP	mAb and Nanobody	12–15 equiv; 2–3 h; RT; pH 8.6	12 min; RT; pH 4.5	(129)
	(±)-H3RESCA-maleimide compared with MMA-NOTA	Affibody	1 equiv; site specific; 90 min; RT; pH 7.5	15 min, 37 °C (RESCA) or 100 °C (NOTA); pH 4	(129)

The number of molar equivalents used (chelator-to-antibody ratio) in conjugation reactions is abbreviated as “equiv” and room temperature is abbreviated as RT. Chemical structures of the chelators are presented in Figure .

Figure 2

Chemical structures of main chelators discussed in this review. If applicable, the most common position for conjugation via a linker has been indicated. [18F]AlF has been indicated to illustrate the special coordination geometry of RESCA.

Bifunctional Chelators (BFCs) and Conjugation Strategies

(i) Antibodies are mostly radiolabeled with radiometals via a bifunctional chelator (BFC) consisting of a chelator to coordinate the radiometal and a linker to allow coupling to the antibody

Antibodies are generally modified by either random or site-specific conjugation to lysines using activated carboxylic acids and isothiocyanates or to thiols using maleimides.[14] For details on recent advances in site-specific conjugation methods, click chemistry, and pretargeting strategies, we refer to the reviews of Morais and Ma,[15] Adumeau et al.,[16,17] Meyer et al.,[18] and to the section on “new conjugation strategies” from the review of Wei et al. from 2020.[19] BFCs should be coupled in an inert and stable way, not affecting the protein integrity, immunoreactivity, and in vivo biodistribution of the antibody and avoiding the release of the radionuclide. Several aspects are of importance to guarantee inertness. BFC conjugation conditions should be relatively mild to avoid alteration of the protein integrity.

Among others, the pH of the conjugation reaction mixture is usually kept between 7 and 9, while high reaction temperature (>50 °C) can seriously alter the secondary and tertiary structures of the antibody and should be avoided to preserve antibody integrity. After BFC conjugation, usually a purification step is needed (i.e., via size exclusion) to remove uncoupled chelator molecules and to improve subsequent radiolabeling yields.

(ii) Conjugation to or near the antigen binding domain of the antibody should be avoided to prevent impairment of immunoreactivity

The larger the antibody is, the more lysines are available outside the antigen binding region, thus reducing the chance of impaired immunoreactivity. This becomes more challenging for small antibody derivatives like nanobodies where a site-specific conjugation might be preferred to avoid conjugation of the BFC to an amino acid to or near the complementary-determining regions (CDR) of the antibody. Site-specific coupling harbors the advantage of controlling where the antibody will be modified, thus resulting in a homogeneous product. This approach, however, requires either engineering of antibodies to introduce functional chemical group for BFC coupling or pretreating the antibody before BFC conjugation to obtain reaction sites.

(iii) The number of chelator molecules coupled per antibody molecule should be kept within limits, avoiding alteration of the normal biodistribution of the mAb

It is generally assumed that one to two chelators per antibody on average do not interfere with the normal biodistribution of the antibody. Apart from the number of chelators coupled, various factors can also be responsible for disturbing the pharmacokinetics of the antibody such as the overall charge, isoelectric point, and hydrophilicity of the radiolabeled molecule. Furthermore, the intramolecular positions of the lysine and thiol functions available for conjugation with the BFC are important. While modification on the Fc part of the mAb should be preferred, conjugation to the variable domain should be avoided to exclude impairment of the immunoreactivity of the mAb toward the antigen binding site.

Quality Controls

Radiotracers based on antibodies usually undergo quality control tests directly after radiolabeling and subsequent purification and formulation. Among the release specifications for preclinical and clinical application are radiochemical purity, protein integrity, and maintenance of biological properties such as antigen binding. Furthermore, storage conditions must be established and a shelf life should be set for which those release specifications are proven to be preserved.

Formulation of radiolabeled antibodies is typically done in aqueous buffers, and quality controls are performed by HPLC (i.e., size exclusion chromatography) or SDS-PAGE using radiodetection to determine the radiochemical purity and protein integrity. TLC or spin filter analysis, based on molecular weight size-exclusion separation, is used to assess the radiochemical purity as well. Finally, binding assays need to be developed to check that the immunoreactivity (binding to the antigen) of the radioimmunoconjugate is preserved via either cell-based or ELISA-like assays.[20] Additionally, production of a radiotracer for clinical use needs to follow Good Manufacturing Practice (GMP) guidelines in a clean and controlled environment with a reproducible and validated manufacturing process and well-defined releasing criteria.[21]

In this review, an overview of radiometal-labeled antibodies that have been evaluated in vivo in the past five years will be given. The constructs selected range from nanobodies to monoclonal antibodies (mAbs) (Figure ) labeled with the often used radiometals 89Zr, 64Cu, and 68Ga; with less common radionuclides 52 Mn, 86Y, 66Ga, and 44Sc; or with 18F using chelated aluminum as the metal. We will discuss practical considerations with respect to the chelators used and strategies for complexation of radiometals as well as the radiolabeling conditions of antibodies with radiometals. Approaches that consist of labeling of chelators first (prelabeling approach), followed by coupling to the biological molecule, are excluded from this review.

Radiometals Evaluated in Immuno-PET

89Zr

Thanks to its half-life of 78.41 h, 89Zr has been extensively used for the radiolabeling of mAbs. 89Zr is readily available via commercial suppliers, produced in a cyclotron via the 89Y(p,n)89Zr nuclear reaction and obtained as 89Zr-oxalate ([89Zr(C2O4)4]4–). 89Zr decays via β+ emission (23%) and electron capture (77%) to 89mY (t1/2 = 15.7 s) and finally to 89Y via γ emission of 909 keV which does not interfere with PET imaging (see Table ).[22]

Remarkably, desferrioxamine (DFO) is still the only chelator used in clinical studies, which can be explained by the fact that it is a well-known chelator that has been used safely for decades as an antidote for iron overload. Furthermore, coupling reactions with commercially available bifunctional derivatives of DFO (TFP-N-suc-DFO and DFO-NCS) have been extensively reported with well-described[23−26] and GMP compliant procedures.[27,28] Recently, some comprehensive reviews have been published by Wei et al.,[29] Yoon et al.,[30] and van Dongen et al.[2] on the preclinical and clinical use of 89Zr-immuno-PET.

Zirconium-89 is typically present in solution as Zr4+, and is a hard Lewis acid with strong affinity for hard Lewis bases such as oxygen. An octadentate coordination sphere is preferred, while DFO is only able to provide hexadendate coordination. Two additional coordinating ligands are supposed to be needed for optimal stabilization of the 89Zr-complex.[31,32] This has led to suboptimal stability with DFO in preclinical in vivo models and uptake of free 89Zr in bones. Whether this is a topic of concern in the clinical setting, especially regarding bone dosimetry at late time points and possible misdiagnosis in case of bone metastasis, is not clear yet and remains to be investigated.[31−37] Multiple research groups have developed octadentate chelators for 89Zr to solve this issue while keeping synthesis, coupling, and radiolabeling conditions facile and mild. Research in the field on new chelators for 89Zr has been extensively reported in reviews;[9,38−45] see Table and Figure for an overview of bifunctional chelators recently evaluated in a preclinical setting.

One of the newly developed octadentate chelators is DFO*,[46] a derivative of DFO with an additional fourth hydroxamate group in comparison with DFO. DFO*-mAb complexes demonstrated improved stability, in vitro as well as in vivo, with clearly less bone uptake.[47−51] Moreover, DFO*, DFO*-NCS, and DFO*-maleimide have recently become commercially available, which should greatly facilitate clinical translation. In a recent study,[50] DFO* was compared with DFOSq, a chelator introduced by Rudd et al. that also showed promising preliminary results,[52] and DFO*Sq, a hybrid chelator used to better understand the influence of the extra hydroxamate group of DFO* and the Squaramide. In vivo uptake in bones was, however, unfavorable for DFOSq in comparison with the DFO* chelators and density function theory (DFT) measurements demonstrated that DFOSq is actually a seven-coordinate complex and thus not able to fully coordinate 89Zr.[53]

Two other bifunctional chelators, p-SCN-Bn-HOPO (based on 3,4,3-(LI-1,2-HOPO))[37] and DFO-cyclo*-pPhe-NCS[48] have shown promising preclinical results. However, the use of p-SCN-Bn-HOPO is hampered by the rather complicated synthesis of the BFC, and no clinical application has been reported yet.[54] DFO-cyclo*-pPhe-NCS performed equally good as DFO*-NCS in preclinical in vivo studies. Differences have been observed in conjugation efficiency, being less efficient for DFO-cyclo*-pPhe-NCS, and in lipophilicity, being higher for DFO-cyclo*-pPhe-NCS.[48] The fact that DFO-cyclo*-pPhe-NCS is a chiral compound makes this candidate less attractive from the pharmaceutical point of view. Finally, DOTA, a well-known chelator for many radiometals, has also been suggested to be able to fully coordinate and stabilize 89Zr. Because of the high temperature (90 °C) needed for radiolabeling DOTA with 89Zr, a prelabeling approach is required comprising the conversion of 89Zr-oxalate to 89Zr-chloride followed by radiolabeling of DOTA and then coupling to an antibody. However, to the best of our knowledge, 89Zr-DOTA-mAb conjugates have not been evaluated in vivo yet.[55,56] Of note, development of novel candidate chelators for 89Zr remains a very active field as exemplified by the recent introduction of oxoDFO*,[57,58] FSC derivatives,[59] 4HMS,[60] DFO2,[61] PCTA,[62] and NOTA.[62] However, for most of these chelators the suitability for stable, efficient, and inert 89Zr labeling of mAbs still has to be demonstrated, while in vivo evaluation to prove their superiority over DFO and DFO* is lacking.

64Cu

64Cu (t1/2 = 12.7 h) is often considered a radionuclide with a short to intermediate half-life suitable for the labeling of antibody fragments with a medium to short biological half-life such as Fab and single-chain variable fragments (scFv). 64Cu is generally obtained via the bombardment of enriched 64Ni and provided as [64Cu]CuCl2. 64Cu has been considered for its theranostic properties not only because of its intrinsic β– (39.0%) emission along with its β+ (17.9%) but also because 64Cu could be used as a pair with the therapeutic β– emitting radionuclide 67Cu, although applications with the latter are still limited due to its rare and expensive production.[130] To reflect on exciting developments regarding 64Cu radiopharmaceuticals, comprehensive reviews have recently been published.[9,10,22,44,130−137]

Due to the hardness of Cu2+ aliphatic and aromatic amines as well as carboxylates have been widely used in BFCs for antibody labeling. Cu2+ is capable of forming complexes with four to six coordinating ligands, of which macrocyclic chelators containing six coordinating ligands are often preferred because of their increased complex stability. Many chelators have been evaluated over the years;[19,42,44,138,139] however, DOTA remains the preferred chelator for evaluation of antibodies preclinically and clinically. This is surprising taking into account that 64Cu-DOTA demonstrates far from optimal stability in vivo resulting in hepatobiliary excretion of released copper and its transchelation to other proteins such as superoxide dismutase. Despite its suboptimal in vivo performance, DOTA has the advantage that it is commercially available and can be used also for other radiometals, while it has been safely used clinically for decades.[140,141] Over the years, however, NOTA has emerged as a successful successor of DOTA in clinical studies showing improved in vivo stability while allowing fast (1 h or less depending on the protein) and mild labeling conditions (RT to 40 °C, pH ∼ 5–6) for heat-sensitive antibodies (see Table ).[142]In vivo preclinical benchmarking studies comparing chelators are still ongoing and unsurprisingly show that DOTA is the least stable chelator for 64Cu (see Table ).

Table 3

Representative Preclinical Benchmarking Studies with 64Cu Chelators Reported in the Last ∼5 Years

64Cu comparison of chelators	antibody derivatives	conjugation conditions	radiolabeling conditions	conclusion/comment	ref
Cu-NOTA-(NCS)/89Zr-DFO-(NCS)	bivalent scFv-Fc	5 equiv; 1 h; 37 °C; pH 9.0	1 h; 37 °C; pH not indicated	Similar uptake at day 1, 89Zr derivative preferred for later imaging time points (as for mAbs)	(147)
DOTA-(NCS)/NOTA-(NCS)	mAbs	20 equiv; 4 h; 4 °C; pH 8.5	45 min; 38 °C; pH 5	NOTA better chelator with, i.e., reduced liver uptake	(148)
NODAGA-(NHS)/DOTA-(NHS)	Affibodies	3–10 equiv; in DMSO, reaction quenched by TRIS pH 8 (time not indicated)	1 h; 37 °C; DOTA-affibody (pH 7.4) or NODAGA-affibody (pH 6.0)	NODAGA better chelator regarding uptake in normal organs	(149)
NODAGA/NOTA	Affibodies	5 equiv; overnight for NODAGA; 2 h for NOTA; RT; pH not indicated	45 min; 95 °C; pH 5.6	NODAGA complex better regarding uptake in normal organs	(150)
				High radiometabolites in kidneys for both tracers (usual for affibodies)
NODAGA(−NHS)/DOTA(−NHS)	mAb	5–100 equiv; 20 h; 4 °C; in 0.1 M borate buffered saline	30 min (DOTA) or 60 min (NODAGA); RT or 40 °C; pH 5.6	Various reaction parameters. NODAGA more stable in vivo than DOTA derivative, similar tumor uptake and less in normal tissues	(151,152)
		20 equiv; RT 3–4 h then 4 °C overnight; pH 8.3	1 h, 40 °C (DOTA-mAb) or RT (NODAGA-mAb); pH 6	Higher in vivo stability of NODAGA-mAb
TE1PA-(NCS)/NOTA-(NCS)/DOTA-(NCS)/DOTA-(NHS)	mAb	20 equiv; overnight; RT; pH 8.5–8.6	30 min; 40 °C; pH 6–7	TE1PA conjugation 5 times more efficient. Better in vivo stability of TE1PA conjugate against DOTA regarding transchelation especially at late time points	(153,154)
phosphinate chelator (L5(-NCS)) compared with NODAGA-(NCS)	mAb	50 equiv; 24 h; 25 °C; pH 7.9	20 min; 37 °C; pH 5.5	Phosphinate chelator superior especially regarding uptake in normal organs	(155)

Other chelators such as various cyclams have been evaluated but do not always show superior in vivo stability compared to DOTA and NOTA derivatives. In addition, CB-TE2A and TETA chelators are promising but usually require radiolabeling conditions too harsh for sensitive antibodies (i.e., 90 °C), and thus their use is restricted to heat-stable proteins.[143,144] Antibodies radiolabeled with sarcophagine (Sar) derivatives have shown promising in vivo stability in the past with fast (20–30 min) radiolabeling at RT.[145] Research on new sarcophagine BFCs is actively ongoing and very recently showed promising results with trastuzumab.[146] Publications with antibody fragments have also been described for which in contrast to intact mAbs, kidney retention is often observed, which most probably is caused by the positive charge of the 64Cu-chelator resulting in binding to negatively charged glomerulus cells in kidneys.[111]

68Ga

In comparison with previously discussed radiometals such as 89Zr and 64Cu, 68Ga emits relatively high-energy positrons (Eβmax = 1.9 Mev) that can hamper image quality (mean range in water 2.9 mm) (Table ). 68Ga is generally produced via the decay of its mother radionuclide Germanium-68 (t1/2 = 271 d) and has emerged as a practical solution for small-scale PET tracer synthesis due the convenience of having a 68Ge/68Ga generator on site. Its short half-life (t1/2 = 68 min) limits, however, the option of central production for multicenter applications. Nevertheless, 68Ga has become a chelator of choice for radiolabeling of peptides and in recent years for antibody derivatives with a short biological half-life such as nanobodies.

68Ga3+ is considered a relatively hard cation, stable in acidic conditions, preferring hexadentate chelation via nitrogen and oxygen donors. However, 68Ga3+ slowly forms insoluble Ga(OH)3 complexes between pH 3 and 7, which requires complexation kinetics faster than their formation. At physiological pH, soluble [Ga(OH)4]− is formed, which shows slow chelation kinetics. As such, radiolabeling conditions are usually chosen that prevent the formation of Ga(OH)3 or in the presence of trapping reagents (i.e., citrate or acetate).[9]

Typically, 68Ga is eluted from the generator as 68Ga3+ in HCl solution. Over the years, many chelators have been developed, and some recent comprehensive reviews discuss in great detail the labeling conditions, yields, and variety of in vivo applications with 68Ga-radiolabeled peptides and antibody derivatives.[9,156,157] Initially, DOTA was often used for complexation of 68Ga even though high reaction temperatures were needed for good radiolabeling efficiency. However, these relatively harsh conditions are not appropriate for heat-sensitive antibodies. For this reason, the use of DOTA in preclinical (see Table ) and clinical[158−160] studies is mainly restricted to antibodies that can handle high temperatures such as affibodies. As for 64Cu, NOTA is by far the most often used chelator for 68Ga (Table ).[161] NOTA has the advantage over DOTA of allowing radiolabeling at RT, and therefore is suitable for a large range of antibody derivatives.

DFO,[125] HBED,[124] and tris(hydroxypyridinone) THP[162] derivatives have also been used in recent years studies, but in vivo benchmarking studies proving their superiority over NOTA derivatives are still lacking even though those chelators provide attractive radiolabeling conditions.[156]

Other Radionuclides

52Mn

52Mn is an interesting emerging radiometal for immuno-PET because of its half-life and low positron energy (t1/2 = 5.59 d, β+ = 29.6%, E(β+)max = 0.576 MeV, Table ) that offers favorable resolution for imaging (in the range of 18F). There are safety concerns because of the concomitant high-energy gammas (744 keV (90%), 935 keV (95%), and 234 and 1434 keV (100%)) that result in a high radiation burden in in vivo applications. 52Mn is commonly produced in a cyclotron via the 52Cr(p,n)52Mn reaction. Manganese is a hard transition metal present in solution mainly in the active oxidation states 2+ and 3+, thus offering complexation as Mn2+ with polyaminocarboxylic acid chelators like DOTA.

Stable Mn has been of particular interest for MRI applications with dual-modality manganese-enhanced magnetic resonance imaging (MEMRI), but despite favorable physical characteristics, its use has been limited, most probably due to the biological toxicity of bulk manganese, its accumulation in organs such as the pancreas, and its neurotoxicity that can lead to Parkinson-like impairments.[163−166] This is certainly not an issue for 52Mn PET tracer applications where the amounts of Mn needed (in the nanomolar range) to obtain a PET signal are very low, thus reducing toxicity issues to the minimum.[167]

To the best of our knowledge, there is only one study that reported on the in vivo evaluation of a 52Mn-labeled mAb. To this end, the anti-CD105 mAb TRC105 was conjugated with DOTA-NCS, followed by radiolabeling at pH 4.5 to 7.5 and a temperature ranging from RT to 55 °C. In vivo data showed a maximum tumor uptake of 19%ID/g 24 h p.i. of the tracer with a slow blood clearance over time and relatively high bone and spleen uptake (>10%ID/g, 120 h p.i.), which requires further investigation.[167]

86Y

Yttrium-86 (t1/2 = 14.74 h, β+ = 33%, 3.1 MeV, Table ) has generated interest as a surrogate isotope of yttrium that could be used as a matching pair for targeted radionuclide therapy with the β–-emitter yttrium-90 (t1/2 = 64.1 h).[168]86Y is most commonly produced via the 86Sr(p,n)86Y reaction. 86Yttrium is a transition metal ion that prefers an octadentate coordination in its 86Y3+ state and has mainly been used with the well-known polyaminocarboxylates chelators, DTPA and DOTA.[169] Just a couple of publications have described the use of 86Y-DTPA radiolabeled antibodies in the last five years with successful radiolabeling under mild conditions (1 h at 37 °C in sodium acetate) including therapy studies with the corresponding 90Y derivatives.[170,171]86Y presents, however, more than 65% of its decay along with γ-rays ranging from 200 to 3000 keV that hamper contrast and quantification with the presence of false coincident γ detection.

66Ga

Gallium-66 (t1/2 = 9.49 h, β+ = 56.5%, 4.2 MeV, Table ) is another β+-emitting isotope of gallium produced by the 66Zn(p,n)66Ga nuclear reaction that is envisaged as a novel tool for PET imaging and has already been applied to peptides.[172]In vivo applications to antibodies are, however, still limited. An anti-EGFR affibody has been conjugated with DFO, followed by radiolabeling with 66Ga, 68Ga, and 89Zr and their in vivo behavior compared. The longer half-life of 66Ga allowed imaging at later time points which was beneficial for the tumor-to-organ ratios.[173] Although 66Ga seems to be a more interesting radionuclide than 68Ga for the labeling of antibody derivatives with a longer serum half-life, application is limited due to its high positron range (Rmean = 9.3 mm in water) and co-emitting gammas that hamper spatial resolution and reliable PET quantification.[174]

44Sc

44Sc can be obtained via a 44Ti/44Sc generator or via production in a cyclotron using a 44Ca(p,n)44Sc reaction (see Table ). Thus far, focus has been on improving the 44Sc production process to reduce costs that has been limiting its applicability.[9,175−179] A half-life of about 4 h and a decay to nontoxic Ca make 44Sc an interesting radionuclide for immuno-PET imaging. Scandium is present in aqueous solutions as the hard trivalent cation Sc3+ with a strong preference for hard oxygen donor ligands and a flexible coordination number (between 3 and 9, with 6 being the most reported).[180]44Sc complexation strongly depends on pH, with pH 4 being considered the most optimal for radiolabeling.[180]44Sc is a PET radionuclide with growing interest in recent years, however, mostly in the field of peptide labeling. This is probably due to the relatively harsh radiolabeling conditions not being compatible with antibodies. To date, first in human studies have been reported with peptides (i.e., PSMA and DOTATOC) but not with larger biomolecules.[181] Moreover, 44Sc is considered to form a theranostic pair with the therapeutic radionuclides 177Lu (t1/2 = 6.7 days) and 47Sc (t1/2 = 3.4 days), although their half-lives do not match optimally.[182]

Various chelators have been tested for 44Sc, with DOTA being favorable, but its application is still restricted to affibodies which can handle the required high reaction temperatures of 90–110 °C.[183,184] Other chelators include a DTPA derivative, which was coupled to a Fab fragment of cetuximab and further evaluated in vivo. Radiolabeling was fast (30 min) at RT and gave better yields than the DOTA and NOTA derivatives.[185] Finally, H3 mpatcn (see Figure ), a small triazamacrocyclic picolinate-functionalized chelator, showed efficient labeling of peptides at RT, but does not seem to have been applied to antibody derivatives yet.[186] Of note, uncoupled Sc as in Sc(III)-citrate has been found to accumulate in liver, spleen, and bone with possible accumulation of colloids in lungs and heart.[180]

18F in [18F]AlF

In this review on radiometals for immuno-PET, it appears relevant to also discuss the progress on labeling of antibodies with 18F via aluminum. 18F is by far the most often used PET radionuclide for small molecule imaging thanks to its short half-life (110 min) and ideal physical properties (Eβmax = 0.6 MeV, mean range in water = 0.6 mm). Labeling procedures with 18F can, however, be time-consuming and harsh with multiple drying and purification steps that decrease overall yields significantly, while the use of organic solvents and high-temperature conditions are not compatible with antibodies. As fluorine is able to form strong bonds with metals, especially aluminum,[187] [18F]AlF radiochemistry is opening new possibilities for fast metal complexation and thus radiolabeling of antibodies.[188−191]

18F is easily available via commercial suppliers and produced in a cyclotron via the 18O(p,n)18F nuclear reaction. AlCl3 is used to prepare a stock solution of Al3+ in typically 0.1 M sodium acetate at pH ∼ 4 to which fluorine is further added and incubated to form a strong [18F]AlF bond followed by purification, for example, over a QMA cartridge to remove free 18F and other metal contaminants.[192] Radiolabeling of NOTA and NODA derivatives with [18F]AlF is done at high temperature (100–110 °C) in a short reaction time (15 min) and results in very stable complexes. However, the high radiolabeling temperature has prevented direct labeling of heat-sensitive antibodies.[193−196] One of the most promising developments in this field is the development of the acyclic pentadentate chelator (±)-H3RESCA which possesses a N2O3 coordinating set of donor atoms, which can strongly complex [18F]AlF at room temperature for which Cleeren et al.[197] filed a patent in 2016.[198,199]

Twelve-to-fifteen times excess of the chelator (±)-H3RESCA, a pentadentate ligand with N2O3 donor groups, is typically reacted with the antibody derivative (i.e., a nanobody) in aqueous solution and incubation for 2–3 h at RT and at pH 8.5–8.7 resulting in a chelator-to-protein ratio between 1 and 2.[198] Radiolabeling is performed under mild conditions (pH 4–5, RT to 37 °C, 12–15 min, see Table ), and a cosolvent (i.e., ethanol) is generally used to increase radiolabeling yields.

18F-labeling of antibodies via this chelator has, however, raised a few points of concern. Elevated bone uptake has been observed indicating demetalation and/or defluorination and seems to depend on the pharmacokinetics of the molecule itself but could also be animal-species-dependent.[198] The assumed mechanism of in vivo degradation involves glomerular filtration and conjugate degradation resulting in release and recirculation of free 18F. This phenomenon might also be dependent on the biological molecule itself, its half-life, preference for renal excretion, and the preclinical model used. Furthermore, the slightly more lipophilic nature of (±)-H3RESCA in comparison with other chelators such as NOTA could induce increased hepatic clearance.[198]In vivo preclinical and clinical studies should confirm the potential of (±)-H3RESCA for [18F]AlF radiolabeling of antibodies.

Conclusion

Radiolabeling of antibodies with radiometals is a growing and exciting field within PET imaging for which tremendous efforts have been made to develop general conjugation and radiolabeling methods with chelators that could be used for multiple radiometals. As illustrated in this review, there is, however, still no ideal chelator that could be used in an optimal way for multiple radiometals and compromises have to be made between ideal and practical aspects. Ideally, such a chelator should be commercially available and able to complex in a stable way multiple radiometals following well-established procedures and without altering the properties of the antibody to boost clinical applications. This remains, however, a challenge for the future of immuno-PET, as each radiometal possesses its own chemical and (sometimes harsh) labeling properties along with its specific decay characteristics. Bioconjugation and radiolabeling procedures should furthermore be GMP-compliant to facilitate translation to the clinic. Production costs and availability of the radiometal, the quality and in vivo performance of the obtained radioimmunoconjugate, and the logistics of its production and distribution are thus primordial parameters to take into account. For those reasons, despite very active developments in preclinical studies, the commercially available chelators, DFO, DOTA, and NOTA, are still by far the most often used chelators in the clinic. Among the other promising radiometal–chelator pairs discussed in this review, future preclinical and clinical studies will confirm their potential as new gold standards for immuno-PET imaging.