Leveraging a Dual Variable Domain Immunoglobulin to Create a Site-Specifically Modified Radioimmunoconjugate

Abstract

Site-specifically modified radioimmunoconjugates exhibit superior in vitro and in vivo behavior compared to analogues synthesized via traditional stochastic methods. However, the development of approaches to site-specific bioconjugation that combine high levels of selectivity, simple reaction conditions, and clinical translatability remains a challenge. Herein, we describe a novel solution to this problem: the use of dual-variable domain immunoglobulins (DVD-IgG). More specifically, we report the synthesis, in vitro evaluation, and in vivo validation of a ¹⁷⁷Lu-labeled radioimmunoconjugate based on ^HER2DVD, a DVD-IgG containing the HER2-targeting variable domains of trastuzumab and the catalytic variable domains of IgG h38C2. To this end, we first modified ^HER2DVD with a phenyloxadiazolyl methlysulfone-modified variant of the chelator CHX-A″-DTPA (PODS-CHX-A″-DTPA) and verified the site-specificity of the conjugation for the reactive lysines within the catalytic domains via chemical assay, MALDI-ToF mass spectrometry, and SDS-PAGE. The chelator-bearing immunoconjugate was subsequently labeled with [¹⁷⁷Lu]Lu³⁺ to produce the completed radioimmunoconjugate — [¹⁷⁷Lu]Lu-CHX-A″-DTPA_PODS-^HER2DVD — in >80% radiochemical conversion and a specific activity of 29.5 ± 7.1 GBq/μmol. [¹⁷⁷Lu]Lu-CHX-A″-DTPA_PODS-^HER2DVD did not form aggregates upon prolonged incubation in human serum, displayed 87% stability to demetallation over a 7 d incubation in serum, and exhibited an immunoreactive fraction of 0.95 with HER2-coated beads. Finally, we compared the pharmacokinetic profile of [¹⁷⁷Lu]Lu-CHX-A″-DTPA_PODS-^HER2DVD to that of a ¹⁷⁷Lu-labeled variant of trastuzumab in mice bearing subcutaneous HER2-expressing BT-474 human breast cancer xenografts. The in vivo performance of [¹⁷⁷Lu]Lu-CHX-A″-DTPA_PODS-^HER2DVD matched that of ¹⁷⁷Lu-labeled trastuzumab, with the former producing a tumoral activity concentration of 34.1 ± 12.1 %ID/g at 168 h and tumor-to-blood, tumor-to-liver, and tumor-to-kidney activity concentration ratios of 10.5, 9.6, and 21.8 respectively at the same timepoint. Importantly, the DVD-IgG did not exhibit a substantially longer serum half-life than the traditional IgG despite its significantly larger size (202 kDa for the former vs. 148 kDa for the latter). Taken together, these data suggest that DVD-IgGs represent a viable platform for the future development of highly effective site-specifically labeled radioimmunoconjugates for diagnostic imaging, theranostic imaging, and radioimmunotherapy.

Graphical Abstract

INTRODUCTION

Over the last three decades, radiolabeled antibodies have become vital tools in oncology for diagnostic imaging, theranostic imaging, and targeted radionuclide therapy (i.e. radioimmunotherapy). Paradoxically, the overwhelming majority of these agents designed to facilitate precision medicine are synthesized in a surprisingly imprecise ways: the random attachment of bifunctional chelators or prosthetic groups to lysines distributed throughout the antibody.^1,2,3 While this methodology is simple and inexpensive, it can interfere with the in vitro and in vivo performance of radioimmunoconjugate in two ways. First, the accidental attachment of cargoes within the complementarity-determining regions of the antibody can harm the immunoglobulin’s affinity and selectivity for its antigen.⁴ And second, stochastic modification methods inevitably produce a heterogeneous mixture of products, each with slightly different biophysical and pharmacophysical properties.^4,5 Taken together, these issues complicate the reproducible synthesis of immunoconjugates and — even more seriously — can yield radioimmunoconjugates with suboptimal in vivo performance.^6–8

To overcome these challenges, a great deal of work has been dedicated to developing site-specific approaches to bioconjugation.⁹ A wide variety of promising methods have emerged, including strategies based on chemoenzymatic transformations^10,11, the modification of reduced disulfide bridges¹², the manipulation of the heavy chain glycans¹³, and the incorporation of ‘clickable’ unnatural amino acids¹⁴. Yet striking a balance between ease, expense, and specificity has proven challenging. To wit, the use of thiol-reactive probes to modify free thiols created via the reduction of interchain disulfide bridges is facile and inexpensive; however, this method can still produce a mixture of regioisomers given the presence of as many as 8 free thiols after reduction¹⁵, and the ‘gold-standard’ reagent for thiol-specific bioconjugations — i.e. the maleimide — forms thioether bonds with cysteines that are unstable in vivo.^16,17,18 In contrast, the chemoenzymatic manipulation of the heavy chain glycans produces highly stable and homogeneous immunoconjugates, but its use of bacterially-derived enzymes represents a challenge from the standpoint of GMP-grade clinical production.¹³ Clearly, the field as a whole is still waiting for an approach to site-specific bioconjugation that will overcome the stubborn inertia of the entrenched yet suboptimal stochastic methods.^19,20

In this work, we present a novel approach to the production of site-specifically modified radioimmunoconjugates: the use of dual variable domain immunoglobulins (DVD-IgG)²¹. The humanized murine antibody h38C2 contains a pair of natural, highly reactive Lys99 residues within the V_H regions of its heavy chains (Figure 1A). These lysines sit within hydrophobic pockets and are deprotonated at physiological pH, making them nucleophilic enough to catalyze aldol and retro-aldol reactions (the source of h38C2’s ‘catalytic antibody’ moniker).^22,23 These nucleophilic lysines have also been shown to react with reagents bearing phenyloxadiazolyl methyl sulfones, functional groups that rapidly form irreversible linkages with free thiols but do not react with normal primary amines.²⁴ This unique reactivity presents an enticing opportunity from a bioconjugation perspective, but these singular lysines exist only in h38C2 and not in other mAb.^22,24–26 How, then, to harness them as platforms for site-specific bioconjugation? The solution lies in the generation of DVD-IgG, 200 kDa engineered immunoglobulins that contain the variable domains of two different mAb alongside constant regions (Figure 1A). In the context of a tumor-targeting immunoconjugate, the DVD-IgG would contain the variable domains of both h38C2 and a cancer antigen-binding mAb.²⁶ The former would be leveraged to facilitate site-specific bioconjugation, while the latter would be depended upon for tumor targeting. This approach is conceptually similar to the use of unnatural amino acids (UAA) but without the expense or complexity of the large-scale production of a UAA-bearing mAb. In 2017, Nanna et al. prepared three separate DVD-IgG that reliably and selectively targeted HER2, CD138, and CD79B in models of breast cancer, multiple myeloma, and non-Hodgkin lymphoma, respectively.^26,27 This system was also used to prepare an antibody-drug conjugate (ADC) with the tubulin inhibitor mono-methyl auristatin F that showed selective cytotoxicity in HER2-expressing cell lines.^24,28

Figure 1.

(A) Schematic of a DVD-IgG with antigen-targeting variable domains in purple, catalytic variable domains in blue, constant domains in gray, and reactive lysines in red; (B) Structure of PODS-CHX-A″-DTPA; (C) Schematic of the reaction between CHX-A″-DTPA-PODS and the nucleophilic lysines of ^HER2DVD; (D) Schematic depicting the modification of ^HER2DVD with CHX-A″-DTPA-PODS and its subsequent labeling with [¹⁷⁷Lu]Lu³⁺.

Our goal for this investigation was to explore the potential of DVD-IgG as scaffolds for nuclear medicine. This approach to bioconjugation is highly modular — i.e. any methyl sulfone-bearing cargo could be used to modify the Lys99 residues, and any mAb could be mined for the second set of variable domains — so our first order of business was designing a model system.²⁴ To this end, we turned to a DVD-IgG containing the catalytic variable domains of h38C2, the antigen-binding variable domains of trastuzumab, and the constant regions of human IgG₁ (^HER2DVD, Figure 1A).²⁹ We chose to modify ^HER2DVD with a derivative of the acyclic chelator CHX-A″-DTPA modified with a phenyloxadiazolyl methyl sulfone (PODS) moiety — PODS-CHX-A″-DTPA¹⁷ — and subsequently label the immunoconjugate with the β-emitting radiometal lutetium-177 (¹⁷⁷Lu; t_1/2 ~ 6.6 d), which has been used in a wide variety of preclinically and clinically validated radioimmunotherapeutics.³⁰ Finally, we performed our in vivo validation experiments using HER2-expressing BT-474 human breast carcinoma cells and employed a ¹⁷⁷Lu-labeled variant of trastuzumab for comparisons with a mAb-based control.³¹ Ultimately, we found that [¹⁷⁷Lu]Lu-CHX-A″-DTPA_PODS- ^HER2DVD exhibited excellent in vitro behavior and, despite its larger size, matched the in vivo performance of [¹⁷⁷Lu]Lu-CHX-A″-DTPA-trastuzumab in a subcutaneous xenograft model of breast cancer.

RESULTS AND DISCUSSION

The first step in the investigation was the creation of our core molecular components. To this end, ^HER2DVD was expressed in Expi293F cells and purified via affinity chromatography. In parallel, PODS-CHX-A″-DTPA was synthesized via the straightforward combination of a PODS synthon bearing an amine-terminated, hydrophilic oligo(ethylene glycol) linker and the amine-reactive bifunctional chelator p-SCN-Bn-CHX-A″-DTPA (Figure 1B; see Supporting Information for synthetic details).

The bioconjugation of PODS-CHX-A″-DTPA to ^HER2DVD was achieved via a one-pot, 3 h incubation of the two components in PBS at pH 7.8. Two variables were explored to optimize the reaction: the molar ratio of the bifunctional chelator to ^HER2DVD (5:1 vs. 10:1) and the temperature of the incubation (RT vs. 37 °C). The progress of the bioconjugation reaction under these conditions was monitored using an assay that follows the ability of the two catalytic lysine residues to convert methodol to a fluorescent aldehyde (Figure 2A). This technique clearly demonstrated that the bioconjugation best proceeded at a molar ratio of 10:1 and a temperature of 37 °C, a combination of conditions at which 90% of the catalytic lysine residues were occupied. The novel immunoconjugate was further characterized via SDS-PAGE and mass spectrometry. ESI-MS further supported the site-specificity of the bioconjugation reaction, revealing the attachment of a single chelator to each of the heavy chains — but not the light chains — of the immunoglobulin (Figure 2B). Both reducing and non-reducing SDS-PAGE displayed increases in the molecular weight of CHX-A″-DTPA_PODS- ^HER2DVD compared to its unmodified parent, with the latter suggesting that this increase in mass was (as expected) limited to the ~65 kDa heavy chain of the immunoglobulin (Figure 2C). Finally, MALDI-ToF mass spectrometry further confirmed the degree of labeling data obtained via methodol assay and ESI-MS, indicating a degree of labeling of 2.3 ± 0.2 chelators/IgG (Figure S1). The fact that this value slightly exceeds the hypothetical maximum of 2 chelator/IgG may be an experimental artifact or, less likely, may stem from the presence of other lysines in ^HER2DVD with slightly increased nucleophilicity.

Figure 2.

(A) Monitoring the progress of the ^HER2DVD bioconjugation reaction using an assay that tracks the reactive lysine-catalyzed retro-aldol degradation of methodol to a fluorescent aldehyde (RFU, relative fluorescent units; mean ± SD of duplicates); (B) Assessing the modification of the heavy (HC) and light chains (LC) of CHX-A″-DTPA_PODS-^HER2DVD synthesized at RT (left) and 37 °C (right) by ESI-MS (DAR = drug-to-antibody ratio); (C) Non-reducing (left) and reducing (right) SDS-PAGE comparing ^HER2DVD (1) and CHX-A″-DTPA_PODS-^HER2DVD (2); (D) Reducing SDS-PAGE (left) of [¹⁷⁷Lu]Lu-CHX-A″-DTPA_PODS-^HER2DVD (1), [¹⁷⁷Lu]Lu-CHX-A″-DTPA-trastuzumab (2), CHX-A″-DTPA_PODS-^HER2DVD (3), and CHX-A″-DTPA-trastuzumab (4). Autoradiography of the same gel (right) revealed radioactivity only associated with the heavy chain of [¹⁷⁷Lu]Lu-CHX-A″-DTPA_PODS-^HER2DVD (1) but with the heavy and light chains of [¹⁷⁷Lu]Lu-CHX-A″-DTPA-trastuzumab (2).

In the next step of the study, CHX-A″-DTPA_PODS- ^HER2DVD was radiolabeled (n = 3) with [¹⁷⁷Lu]LuCl₃ under standard conditions: 1 h at 37° C in 0.25 M ammonium acetate buffer (pH 5.5). After purification via gel filtration chromatography, the final radioimmunoconjugate — [¹⁷⁷Lu]Lu-CHX-A″-DTPA_PODS-^HER2DVD — was isolated in ~81% radiochemical yield and a specific activity of 29.5 ± 7.1 GBq/μmol.³² The stability of this radioimmunoconjugate was assayed via incubation in human serum for 168 h at 37 °C. Over this period, radio-iTLC analysis revealed that [¹⁷⁷Lu]Lu-CHX-A″-DTPA_PODS-^HER2DVD remained >87% intact with respect to the demetallation of [¹⁷⁷Lu]Lu³⁺, while size exclusion HPLC indicated that the radioimmunoconjugate did not produce aggregates to an appreciable degree (Figures S4 and S5).

Before moving on to the in vitro characterization of [¹⁷⁷Lu]Lu-CHX-A″-DTPA_PODS-^HER2DVD, we first wanted to create a trastuzumab-based standard to which we could compare the novel radioimmunoconjugate. To this end, we employed the commercially available 150 kDa mAb trastuzumab with the same chelator (CHX-A″-DTPA) and the same radiometal (¹⁷⁷Lu). However, the absence of catalytic lysines created a conundrum for bioconjugation. We could have reduced the mAb and modified its free thiols with PODS-CHX-A″-DTPA, but this would have led to the comparison of one construct labeled via cysteines to another labeled via lysines. We could have site-specifically modified trastuzumab via the manipulation of the heavy chain glycans, but this would have led to the comparison of one construct with intact glycans to another with truncated sugar chains. In the end, some sort of “apples to oranges” situation was inevitable, so we simply modified trastuzumab in a stochastic manner with p-SCN-Bn-CHX-A″-DTPA since we were primarily interested in comparing the pharmacokinetic profiles of the ~150 kDa mAb with the ~200 kDa ^HER2DVD. MALDI-ToF mass spectrometry revealed that the immunoconjugate — CHX-A″-DTPA-trastuzumab — boasted a degree of labeling of 1.7 ± 0.5 chelators/mAb. And after radiolabeling (n = 3), [¹⁷⁷Lu]Lu-CHX-A″-DTPA-trastuzumab was isolated in 87% radiochemical yield and a specific activity of 21.3 ± 4.3 GBq/μmol. Both radioimmunoconjugates were analyzed via SDS-PAGE autoradiography (Figure 2D). As expected, [¹⁷⁷Lu]Lu-CHX-A″-DTPA_PODS-^HER2DVD was radiolabeled specifically on the heavy chain, while radioactivity was associated with both the heavy and light chains of the stochastically labeled [¹⁷⁷Lu]Lu-CHX-A″-DTPA-trastuzumab. The in vitro binding of both [¹⁷⁷Lu]Lu-CHX-A″-DTPA_PODS-^HER2DVD and [¹⁷⁷Lu]Lu-CHX-A″-DTPA-trastuzumab were assayed in triplicate using magnetic beads functionalized with streptavidin and decorated with recombinant HER2³³. Both showed high levels of immunoreactivity: 95 ± 4 % for the former and 98 ± 0² % for the latter (Table 1). These values dropped to ~25% in a blocking experiment with unlabeled “cold” immunoglobulin and <5% with “naked” beads without HER2 (Figure S3).

Table 1.

Comparison of [¹⁷⁷Lu]Lu-CHX-A′′-DTPA_PODS-^HER2DVD and [¹⁷⁷Lu]Lu-CHX-A′′-DTPAtrastuzumab.

Platform	MW (kDa)	DTPA/IgG	Molar Activity (GBq/μmol)	Immunoreactivity (%)	Serum Half-Life (h)	%ID/g Blood (168 h)	%ID/g Tumor (168 h)
HER2DVD	200	2.3 ± 0.2	29.5 ± 7.1	95.4 ± 3.8	87	3.3 ± 2.3	34.1 ± 12.1
trastuzumab	150	1.7 ± 0.5	21.3 ± 4.3	98.4 ± 0.2	77	4.7 ± 2.5	33.6 ± 12.7

The most important experiment in the investigation is — of course — the comparison of the in vivo behavior of [¹⁷⁷Lu]Lu-CHX-A″-DTPA_PODS-^HER2DVD to that of [¹⁷⁷Lu]Lu-CHX-A″-DTPA-trastuzumab. To this end, biodistribution experiments were performed using athymic nude mice bearing HER2-expressing BT-474 human invasive ductal breast carcinoma xenografts. The mice (n = 5 per time point) were first injected with either [¹⁷⁷Lu]Lu-CHX-A″-DTPA_PODS-^HER2DVD [1.0–1.2 MBq (122 MBq/mg) in 100 μL of 1´ PBS] or [¹⁷⁷Lu]Lu-CHX-A″-DTPA-trastuzumab [0.5–1.3 MBq (130 MBq/mg) in 100 μL of 1´ PBS]. After 24, 72, 120, or 168 h, the mice of each cohort were euthanized; their tumors and relevant tissues were harvested, washed, dried, and weighed; and the amount of radioactivity in each tissue was measured using a gamma counter calibrated for ¹⁷⁷Lu.

The biodistribution data clearly revealed the promising in vivo performance of [¹⁷⁷Lu]Lu-CHX-A″-DTPA_PODS-^HER2DVD (Figure 3 and Table S1). At only 24 h post-injection, the radioimmunoconjugate produced a tumoral activity concentration of 17.5 ± 11.1 %ID/g, a number that increased to a maximum of 38.8 ± 9.2 %ID/g at 120 h p.i. Just as importantly, the levels of uptake and retention in healthy tissues remained low. To wit, the background tissues with the highest level of accretion other than the blood (vide infra) were the liver and ovaries. In the former, the radioimmunoconjugate produced activity concentrations of 3.7 ± 1.2 %ID/g, 3.4 ± 1.3 %ID/g, and 3.5 ± 2.1 %ID/g at 24, 120 and 168 h, respectively. In the latter, [¹⁷⁷Lu]Lu-CHX-A″-DTPA_PODS-^HER2DVD produced uptake values of 5.7 ± 1.3 %ID/g, 3.3 ± 0.6 %ID/g, and 2.2 ± 1.5 %ID/g at the same time points. Critically, the uptake values for the DVD-based radioimmunoconjugate were remarkably similar to the activity concentrations produced by [¹⁷⁷Lu]Lu-CHX-A″-DTPA-trastuzumab (Figure 3, Table S1, and Table S2). Indeed, no statistically significant differences were observed in either the tumors or healthy background tissues. The serum half-lives of [¹⁷⁷Lu]Lu-CHX-A″-DTPA_PODS-^HER2DVD and [¹⁷⁷Lu]Lu-CHX-A″-DTPA-trastuzumab were described by a multi-day exponential clearance model based on %ID/g measurements at 24, 72, 120, and 168 h. The serum half-lives of the two radioimmunoconjugates were similar despite the larger size of the DVD-IgG: ~87 h for [¹⁷⁷Lu]Lu-CHX-A″-DTPA_PODS-^HER2DVD and ~77 h for [¹⁷⁷Lu]Lu-CHX-A″-DTPA-trastuzumab (Figure S5, Table S4).

Figure 3.

Biodistribution data acquired 24, 72, 120, and 168 h after the administration of either [¹⁷⁷Lu]Lu-CHX-A″-DTPA_PODS-^HER2DVD 1.0 – 1.2 MBq (122 MBq/mg) in 100 μL of PBS] or [¹⁷⁷Lu]Lu-CHX-A″-DTPA-trastuzumab [0.5 – 1.3 MBq (130 MBq/mg) in 100 μL of PBS] to female athymic nude mice bearing subcutaneous BT-474 human breast cancer xenografts. The data are presented as %ID/g ± S.D. The values for the stomach, small intestine, and large intestine include contents.

Radiation dose rates and therapeutic indices for both radioimmunoconjugates were calculated from the biodistribution data (Figure 3, Table 2, Tables S1, and S2). Not surprisingly given the biodistribution data, the dosimetry data are similar for both radioimmunoconjugates. For example, the tumor absorbed dose for [¹⁷⁷Lu]Lu-DTPA-A″-CHX_PODS-^HER2DVD is 6.27 mGy/kBq, while that of [¹⁷⁷Lu]Lu-CHX-A″-DTPA-trastuzumab is 6.61 mGy/kBq. In both cases, the compartment with the second highest absorbed dose is the blood, with a value of 0.92 mGy/kBq for the former and 1.39 mGy/kBq for the latter. For many tissues, the therapeutic indices of [¹⁷⁷Lu]Lu-CHX-A″-DTPA_PODS-^HER2DVD appear slightly higher than those of [¹⁷⁷Lu]Lu-CHX-A″-DTPA-trastuzumab, but it is unlikely that these values cross the line into statistical significance.

Table 2.

Dosimetry calculations for [¹⁷⁷Lu]Lu-CHX-A′′-DTPA_PODS-^HER2DVD and [¹⁷⁷Lu]Lu-CHX-A′′-DTPA-trastuzumab in female athymic nude bearing subcutaneous HER2-positive BT-474 ductal breast carcinoma xenografts.

	[177Lu] Lu-CHX-A′′-DTPAPODS-HER2DVD		[177Lu]Lu-CHX-A′′-DTPA-trastuzumab
Organ	Absorbed Dose (mGy/kBq)	T.I.	Absorbed Dose (mGy/kBq)	T.I.
Tumor	6.3	-	6.6	-
Blood	0.9	6.8	1.4	4.8
Heart	0.2	28.4	0.3	23.3
Lungs	0.3	22.1	0.5	13.0
Liver	0.8	8.3	0.5	13.6
Spleen	0.3	20.3	0.3	26.3
Pancreas	0.1	66.8	0.1	57.3
Stomach	0.1	68.3	0.1	55.5
Small Intestine	0.1	46.6	0.1	50.5
Large Intestine	0.1	43.9	0.1	48.5
Kidneys	0.3	23.8	0.3	21.8
Muscle	0.0	149.9	0.1	101.5
Bone	0.3	24.7	0.1	58.6
Ovaries	0.5	11.9	0.6	10.7
Carcass	0.2	25.9	0.3	24.0

CONCLUSION

Taken together, the chemical, in vitro, and in vivo data from this investigation underscore the promise of DVD-IgG as platforms for diagnostic and therapeutic radioimmunoconjugates. The unique biochemical properties of ^HER2DVD facilitated the creation of a homogeneous, stable, and well-defined immunoconjugate. The use of the catalytic lysines within DVD-IgG to facilitate site-specific bioconjugation offers several advantages over other extant methods: it does not require the reduction of the immunoglobulin, eschews the involvement of bacterial enzymes, and avoids the complexity and expense of genetic code expansion. We would be remiss not to concede, however, that this approach does require antibody engineering and (at present) inevitably produces an immunoconjugate with a molecular weight of 200 kDa (vide infra).

While an optimal outcome would have been the [¹⁷⁷Lu]Lu-CHX-A″-DTPA_PODS-^HER2DVD displaying better in vitro and in vivo performance than [¹⁷⁷Lu]Lu-CHX-A″-DTPA-trastuzumab, that outcome was unlikely given that both radioimmunoconjugates contained identical antigen-binding domains. The actual results were encouraging in their own right: the ^HER2DVD-based radioimmunoconjugate produced similar tumoral activity concentrations and tumor-to-background activity concentration ratios to its mAb-based cousin. Particularly notable was that the serum half-life of [¹⁷⁷Lu]Lu-CHX-A″-DTPA_PODS-^HER2DVD did not appear to be dramatically different than that of [¹⁷⁷Lu]Lu-CHX-A″-DTPA-trastuzumab, a somewhat surprising development given the larger size of the former.

Moving forward, we plan to expand our exploration of the applications of DVD-IgG in nuclear medicine. First, we are currently working to exploit the orthogonal reactivity of another DVD-IgG — heterodimeric Lys99/Arg99 h38C2 — to create a doubly-labeled immunoconjugate that carries both a toxin and a therapeutic radionuclide.^28,34,35 And second, we recognize the over-arching trend within the field towards the use of smaller (rather than larger) immunoglobulins as vectors for radiopharmaceuticals. Indeed, site-specific labeling is likely to be even more important in the context of antibody fragments given their smaller size. As a result, we are working toward designing antibody fragments such as scFvs (~30 kDa) and single domain antibodies (~15 kDa) that contain the catalytic lysine motif of h38C2 in order to facilitate site-specific bioconjugation^36,37. In the end, we look forward to seeing the impact that this bioconjugation technology could have on the fields of immunoPET, immunoSPECT, and radioimmunotherapy.

Abbreviations

DVD

dual-variable domain

mAb

monoclonal antibody

IgG

immunoglobulin G

ADC

antibody-drug conjugate

ESI-MS

electrospray ionization mass spectrometry

DOL

degree-of-labeling

SDS-PAGE

sodium dodecyl sulfate-polyacrylamide gel electrophoresis

MALDI-ToF

matrix-assisted laser desorption/ionization-time of flight mass spectrometry

Lu

lutetium

CHX-A″-DTPA

2,2’-((2-(((1S,2S)-2-(bis(carboxymethyl)amino)cyclohexyl) (carboxymethyl)amino)ethyl)azanediyl)diacetic acid

PODS

N¹-(3-(2-(2-(3-Aminopropoxy)ethoxy)ethoxy)propyl)-N⁴-(4-(5-(methylsulfonyl)-1,3,4-oxadiazol-2-yl)phenyl)succinamide

HER2

human epidermal growth factor receptor 2

%ID/g

% of injected dose per gram

PBS

phosphate-buffered saline

DMSO

dimethylsulfoxide

EDTA

ethylenediaminetetraacetic acid

iTLC

instant thin-layer chromatography

PET

positron emission tomography

SPECT

single photon emission computed tomography