Evaluation of Candidate Theranostics for 227Th/89Zr Paired Radioimmunotherapy of Lymphoma

Visual Abstract

Radi otheranostic agents provide a unique ability to detect, characterize, treat, and monitor sites of disease with exceptional specificity. A persistent challenge for clinical theranostics is the development of suitably matched therapeutic and diagnostic agents that provide correlating pharmacokinetic data to guide therapeutic application. Ideally, this goal is realized in the form of a targeted agent that can be labeled with radionuclides for either imaging or therapy without other chemical changes. Radiometals must be stably bound to a molecularly specific vector (a small molecule, peptide, or antibody) to achieve localized uptake. The extended biologic residency time and longer radiologic half-life (t 1 =2) of isotopes used for antibody-based agents add a requirement for greater stability. To date, a limited number of chelates have been clinically applied, notably from the DOTA and diethylenetriaminepentaacetic acid classes (1). Advancements in radioisotopes available for theranostic applications necessitate radiologic and chemical efforts to achieve stable, safe, and effective radiopharmaceutical preparation.
Interest in treatments using a-particle-emitting isotopes with high linear-energy transfer continues to grow. A promising isotope that has been widely used to date is 225 Ac, yet the supply of isotopically pure 225 Ac is limited (2). Theranostic pairs for 225 Ac radioimmunotherapy typically use 111 In (3,4). Although these isotopes have reasonably close half-lives (2.8 and 9.8 d t 1 =2 for 111 In and 225 Ac, respectively), SPECT imaging presents challenges in image quantification for pharmacokinetics and dosimetry (5). Alternatively, the radiotheranostics of 225 Ac using 89 Zr (t 1 =2, 3.3 d) for PET have highly similar pharmacokinetics; however, different chelators are required for coordination of each isotope (6,7). 227 Th (t 1 =2, 18.7 d) is produced from 227 Ac (t 1 =2, 21.7 y) with a branching ratio of 98.6% and produces 5 a-particles, including from its first daughter, 223 Ra (t 1 =2, 11.4 d) (Supplemental Fig. 1; supplemental materials are available at http://jnm.snmjournals.org). Tetravalent thorium bears a 5f 0 electronic configuration and is typically 8-, 10-, or even 12-fold coordinated. Chelation with 227 Th has been limited to a few bifunctional ligands, such as macrocyclic DOTA (displaying inefficient labeling yields), and newer hydroxypyridinone or picolinic acid constructs. It can be challenging for such ligands to also stably complex PET isotopes (8)(9)(10)(11), as chelation chemistries are often class-specific (transition metals, lanthanides, actinides, or other heavy metals). Cross-class metal radiolabeling involves different chemistries and mechanisms (12,13), and evaluation of singleagent theranostic precursors is ongoing (9).
In this work, we evaluated 4 antibody-chelator conjugates for in vitro and in vivo stability using ofatumumab, a human anti-CD20 antibody (14,15). The most stable 227 Th chelator conjugate, L804, was evaluated in vivo for tumor-targeting capability in Raji tumor-bearing mice. An 89 Zr-L804-theranostic analog was compared, as well as conventional 89 Zr-chelating DFO. Data demonstrate long-term stability and a pharmacokinetic match between 89 Zr tracer and 227 Th radiotherapy, with translational potential for quantitative imaging and treatment.

Radiopharmaceutical Quality Control
Protein concentration was determined by bicinchoninic acid assay, with more than 90% recoveries. Radiochemical yields were calculated as the ratio of initial activity to measured activity obtained after purification, using g-counting and calibrated high-purity germanium (GEM-50195-S; Ametek) detection (for 227 Th).
Radiochemical purity (RCP) evaluation used thin-layer chromatography (AR-2000; Bioscan) and fast protein liquid chromatography (AKTA; GE Healthcare) for both 227 Th and 89 Zr (Supplemental Figs. 6 and 7). Labeled antibodies were migrated on silica-coated paper with an aqueous solution of diethylenetriaminepentaacetic acid (10 mM, pH 5). A control strip of unchelated 227 Th dissolved in NH 4 OAc displayed complete migration to the front of the strip. After thin-layer chromatography reading, samples were bisected for quantitative radioisotopic determination by high-purity germanium. Radioisotopic purity was verified after purification (Supplemental Fig. 8). Stability and purity were determined using fast protein liquid chromatography with ultraviolet light (280 nm) coupled with in-line radiodetection (Lablogic) for 89 Zr, with 1-mL fraction collection for 227 Th/ 223 Ra g-counting.
In Vitro Stability Assay 227 Th-ofatumumab constructs (100 mg/0.74-0.925 MBq) were incubated in human plasma diluted 1:10 at 37 C, under gentle shaking over 1 half-life of 227 Th. To monitor 227 Th dissociation from antibody, samples were surveyed by thin-layer chromatography and size-exclusion chromatography fast protein liquid chromatography every other day. Thin-layer chromatography sections and fast protein liquid chromatography fractions were g-counted (protocol below). 227 Th activities integrated at the antibody retention time (12-14 min) over the sum of eluted activity defines the RCP percentage of 277 Th-ofatumumab.
Immunoreactivity was evaluated by the assay of Lindmo et al. (23). Cells were incubated with the labeled samples (5 ng of 227 Th conjugate, 16.7 6 1.33 kBq) and blocked with unlabeled ofatumumab. Raji cells (12 3 10 6 ) were incubated for 1 h in phosphate-buffered saline and 1% bovine serum albumin and washed, in triplicate.

Administration and In Vivo Distribution
The studies were approved by the Institutional Animal Care and Use Committee. For organ distribution, female 6-to 8-wk-old Swiss Webster mice (Charles River) were intravenously administered constructs through the retroorbital sinus. The animals received 5.55-9.25 kBq of 227 Thlabeled antibody or 740 kBq of 89 Zr analogs. Injections were adjusted with unlabeled precursor to 20 mg of antibody per injection (supplemental materials). At the times indicated, mice were killed by CO 2 asphyxiation and organs were g-counted (Wizard 2 ; Perkin Elmer). 227 Th and 223 Ra (at equilibrium) activities were determined by decomposing the g-spectra, and percentage injected activity (%IA) per gram of tissue for 227 Th was computed. Absolute activity per organ (Bq/g) was defined using a g-counting methodology, applying serial dilutions of a calibrated 223 Ra source and Bateman equation-corrected 227 Th decay spectra (24). injection (R4 microPET; Siemens). A blocking cohort with 200 mg of unlabeled ofatumumab (2 h before tracer) was included. The scanner was calibrated with a mouse-sized cylinder phantom of aqueous 18 F with a known activity concentration (25), energy windows of 350-650 keV, and coincidence timing of 6 ns. Corrected scanner data were reconstructed by an iterative 3-dimensional maximum a priori algorithm. Volumes of interest were defined, and %IA/mL was computed (ASIPro; Siemens).

In Vivo Distribution
Acute 227 Th stability was assessed in vivo with naïve mice at 24 h after injection (Fig. 3). 227 Th-labeled HEHA and DFOcyclo* constructs were insufficiently stable, with elevated liver uptake (both) and spleen uptake (DFOcyclo*) (.20 %IA/g). In contrast, 227 Th-DOTAand 227 Th-L804-ofatumumab presented nearly identical distributions and no significant differences in organ uptake. The concatenated decay of 227 Th (Supplemental Fig. 1) presents opportunities and challenges for drug development. We decomposed 227 Th activities from daughter 223 Ra (at equilibrium) and analyzed 227 Th-L804ofatumumab distribution. 223 Ra does not decay in place, where 227 Th accumulates, but rather recirculates and is sequestered in the skeleton (Figs. 3B and 3C) in agreement with  previous reports (22,27). Initial 227 Th uptake in lungs, liver, and kidney (.10 %IA/g) decreased over 2 wk to no more than 5%IA/g, suggesting clearance and elimination of antibody (Supplemental Fig. 10). Dosimetric evaluation of a 150 kBq/kg treatment was computed to predict human organ-absorbed doses using IDAC-Dose, version 2.1 (Supplemental Table 1) (28)(29)(30). The highest values for bone, kidney, liver, and spleen ranged from 71 to 65 mGy/MBq; heart wall and bone marrow were both 50 mGy/MBq.

Tumor-Targeting Evaluation
The 227 Th-L804 conjugate was selected as the lead agent for further evaluation. We first investigated the tumor-targeting ability of 227 Th-L804-ofatumumab in CD20-positive Raji tumors. Mice were randomized to receive 227 Th-L804-ofatumumab, control 227 Th-L804-IgG, or 227 Th-L804-ofatumumab preceded by unmodified ofatumumab. The early blood signal for the unblocked group (21.6 6 1.9 %IA/g) decreased with time to 7.5 6 1.8 %IA/g at 7 d (Fig. 4). Control IgG uptake was significantly greater in the spleen over the course of the experiment, whereas 227 Th tumor uptake was significantly higher for the targeted construct at all time points (to control IgG, P , 0.01) and to blocked group at 7 d (P , 0.05). Peak tumor uptake at day 3 for 227 Th-L804-ofatumumab achieved 20 6 1 %IA/g (Fig. 4).
The theranostic capability of L804ofatumumab for 89 Zr PET was tested in CD20-positive SU-DHL-6 xenografts (Fig. 6). Recapitulating Raji accumulation of 227 Th-L804-ofatumumab, we observed high-contrast delineation with 89 Zr-L804-ofatumumab and a low skeletal signal. To confirm specificity, blocking antibody was administered to a representative animal; the result was decreased tumor uptake (Supplemental Fig. 11). Metastatic invasion of lymph nodes was also visualized, along with primary tumor, and was confirmed by histologic analysis (Supplemental Fig. 12) (32).

DISCUSSION
Radiopharmaceutical therapy is an emerging cancer treatment class. Tempering enthusiasm are concerns of off-target tissue effects and the limited availability of several radionuclides. a-particle-emitting therapy confronts both concerns because demand for a key radionuclide, 225 Ac, greatly exceeds supply (33). Alternatives include 223 Ra (however, receptor-specific targeting is challenging) (34) and 227 Th (for which there is an ample stock for early-phase trials) (35,36). Labeling with 227 Th has been achieved in the past using DOTA, octapa, and octadentate 3,2-hydroxypyridinonate structures (37), with the last of these used in clinical trials (NCT03507452 and NCT02581878). However, 227 Th lacks suitable in vivo stability with 89 Zr compatible chelators, potentially precluding theranostic use (22,36). Here, we chose 4 chelators from different classes to test 227 Th coordination efficiency, stability, purity, and cancer cell receptor targeting, and we evaluated the lead conjugate as a companion 89 Zr diagnostic.
Chelator selection was based on chemical attributes and prior experience. DOTA is a versatile chelator and has previously been used for 227 Th coordination (22,38), which requires a 2-step procedure (26,39). The result was poor radiolabeling yields (,5%) and low specific activity (0.8 GBq/g). Previously reported 227 Th-DOTA-antibody specific activities exceed the results of this work for 227 Th-DOTA-ofatumumab, suggesting that antibody labeling can be further optimized (39). HEHA is a large cyclic chelator with 12 donor atoms, potentially amenable to Th 41 coordination (40). HEHA is an efficient in vitro chelator for 225 Ac 31 (41) but has limited in vivo applicability (42). Conjugated to ofatumumab, HEHA complexed 227 Th with limited efficiency and RCP, and data indicate that 227 Th-HEHA-ofatumumab lacks in vivo stability.
DFOcyclo*-and L804-ofatumumab presented the most interesting radiolabeling efficiencies, purities, and specific activities with 227 Th, in line with recent reports (43). DFOcyclo* is a linear chelator of 4 hydroxamate donors providing octadentate coordination. It presents features similar to those of DFO, with the addition of a fourth cyclic hydroxamic acid motif for additional stability to complexes embedding an 8-coordinated metal (44). We also investigated a macrocyclic approach using L804 articulated with four 1-hydroxypyridin-2-one chelators. Previously, L804 immune constructs have shown high affinity for 177 Lu and 89 Zr (45) and potential for actinides (46). Both DFOcyclo* and L804 presented attractive in vitro stability, suggesting strong coordinating features for 227 Th formulations. Surprisingly, despite excellent DFOcyclo* in vitro results, the elevated liver and spleen accumulations suggest instability of the conjugate or metal decomplexation. 227 Th-L804-ofatumumab remained intact in vivo, as CD20-expressing tumor recognition was achieved for 2 tumor mouse models of lymphoma. 227 Th-L804-ofatumumab organ distributions were similar to the DOTA conjugate in naïve mice, with extended blood content and low bone uptake. In vitro performance and in vivo utility indicate that L804 is an effective chelator of 227 Th for radiopharmaceutical applications.
g-spectroscopic analysis of the radiolabeled material showed selective 227 Th labeling with insignificant 223 Ra. However, concatenated decay leads to production of daughters over time, complicating quality control and in vivo evaluation (47). 227 Th-L804-ofatumumab was administered with high radionuclidic purity, and in vivo ingrowth of 223 Ra was notable for its skeletal redistribution anticipated from 223 RaCl 2 distribution in mice (27,48) and other 227 Th conjugates (49). 227 Th-L804-ofatumumab organ distribution over 2 wk indicates clearance from off-target organs including lungs, liver, spleen, and kidneys. Predicted human dosimetry showed that bone, kidney, and spleen may receive the highest absorbed doses for activity administrations of 150 kBq/kg. We computed low bone marrow dose estimates (,2 Gy). Considering stable 227 Th coordination, the magnitude of tumor activity-delivery, and dosimetry, 227 Th-L804 may drive further interest in radioimmunotherapy.
Finally, we addressed the theranostic potential for quantitative imaging using 89 Zr-L804-ofatumumab. Subtle but significant differences were measured in blood (early time points) versus 227 Th, and these differences resolved at 2 wk; otherwise, a nearly identical distribution was observed. In contrast, the increasing bone uptake with DFO conjugate indicated inadequate long-term stability. PET imaging of 89 Zr-L804-ofatumumab further confirmed effective chelation of 89 Zr by L804, displaying-with clear contrastprimary tumor SU-DHL-6 and diseased lymph nodes and showing low skeletal uptake.

CONCLUSION
L804 is the most stable and versatile chelator of those tested, providing facile coordination of 227 Th and 89 Zr. Stable chelation of 227 Th was demonstrated and applied for tumor-targeted delivery across 2 lymphoma models. These data support the further development of 227 Th/ 89 Zr antibody theranostics using this chemically identical precursor.

DISCLOSURE
Financial support was received from NIH NCI (R01CA229893, R01CA240711, and R01EB02925901 to Daniel Thorek), P30 CA091842, the Children's Discovery Institute of Washington University in St. Louis and St. Louis Children's Hospital (to Diane Abou), the Centre National de la Recherche Scientifique (CNRS), the Conseil R egional de Bourgogne through the Plan d'Action Regional pour l'Innovation (program PARI II "Pharmaco-imagerie et agents th eragnostiques"), the European Regional Development