Engineered Cells as a Test Platform for Radiohaptens in Pretargeted Imaging and Radioimmunotherapy Applications.

Pretargeted imaging and radioimmunotherapy approaches are designed to have superior targeting properties over directly targeted antibodies but impose more complex pharmacology, which hinders efforts to optimize the ligands prior to human applications. Human embryonic kidney 293T cells expressing the humanized single-chain variable fragment (scFv) C825 (huC825) with high-affinity for DOTA-haptens (293T-huC825) in a transmembrane-anchored format eliminated the requirement to use other pretargeting reagents and provided a simplified, accelerated assay of radiohapten capture while offering normalized cell surface expression of the molecular target of interest. Using binding assays, ex vivo biodistribution, and in vivo imaging, we demonstrated that radiohaptens based on benzyl-DOTA and a second generation "Proteus" DOTA-platform effectively and specifically engaged membrane-bound huC825, achieving favorable tumor-to-normal tissue uptake ratios in mice. Furthermore, [86Y]Y-DOTA-Bn predicted absorbed dose to critical organs with reasonable accuracy for both [177Lu]Lu-DOTA-Bn and [225Ac]Ac-Pr, which highlights the benefit of a dosimetry-based treatment approach.

An N-Acetylgalactosamino Dendron-Clearing Agent for High-Therapeutic-Index DOTA-Hapten Pretargeted Radioimmunotherapy.

Clearing agents (CAs) can rapidly remove nonlocalized targeting biomolecules from circulation for hepatic catabolism, thereby enhancing the therapeutic index (TI), especially for blood (marrow), of the subsequently administered radioisotope in any multistep pretargeting strategy. Herein we describe the synthesis and in vivo evaluation of a fully synthetic glycodendrimer-based CA for DOTA-based pretargeted radioimmunotherapy (DOTA-PRIT). The novel dendron-CA consists of a nonradioactive yttrium-DOTA-Bn molecule attached via a linker to a glycodendron displaying 16 terminal α-thio-N-acetylgalactosamine (α-SGalNAc) units (CCA α-16-DOTA-Y3+; molecular weight: 9059 Da). Pretargeting [177Lu]LuDOTA-Bn with CCA α-16-DOTA-Y3+ to GPA33-expressing SW1222 human colorectal xenografts was highly effective, leading to absorbed doses of [177Lu]LuDOTA-Bn for blood, tumor, liver, spleen, and kidneys of 11.7, 468, 9.97, 5.49, and 13.3 cGy/MBq, respectively. Tumor-to-normal tissues absorbed-dose ratios (i.e., TIs) ranged from 40 (e.g., for blood and kidney) to about 550 for stomach.

Pharmacokinetics and Biodistribution of a [89Zr]Zr-DFO-MSTP2109A Anti-STEAP1 Antibody in Metastatic Castration-Resistant Prostate Cancer Patients.

A six-transmembrane epithelial antigen of prostate-1 (STEAP1) is a newly identified target in prostate cancer. The use of radio-labeled STEAP1-targeting antibodies with positron emission tomography (PET) may allow for detection of sites of metastatic prostate cancer and may refine patient selection for antigen-directed therapies. This was a prospective study in seven patients with metastatic castration-resistant prostate cancer who had at least one archival biopsy that was STEAP1-positive by immunohistochemistry. Patients received intravenous injections of ∼185 MBq and 10 mg of [89Zr]Zr-DFO-MSTP2109A, a humanized IgG1 monoclonal antibody directed against STEAP1. PET/CT images, blood samples, and whole-body counts were monitored longitudinally in six patients. Here, we report on safety, biodistribution, pharmacokinetics, dose estimates to normal tissues, and initial tumor targeting for this group of patients. There was no significant acute or subacute toxicity. Favorable biodistribution and enhanced lesion uptake (in both bone and soft tissue) were observed on imaging using a mass of 10 mg of DFO-MSTP2109A. The best lesion discrimination was seen at the latest imaging time, a median of 6 days postadministration. Pharmacokinetics showed a median serum T1/2 ß of 198 h, volume of central compartment of 3.54 L (similar to plasma volume), and clearance of 19.7 mL/h. The median biologic T1/2 for whole-body retention was 469 h. The highest mean absorbed doses to normal organs (mGy/MBq) were 1.18, 1.11, 0.78, 0.73, and 0.71 for liver, heart wall, lung, kidney, and spleen, respectively. Excellent targeting of metastatic prostate sites in both bone and soft tissue was observed, with an optimal imaging time of 6 days postadministration. The liver and heart were the normal organs that experienced the highest absorbed doses. The pharmacokinetics were similar to other antibodies without major cross-reactivity with normal tissues. A more detailed analysis of lesion targeting in a larger patient population with correlation to immunohistology and standard imaging modalities has been reported.

ImmunoPET Imaging of Endogenous and Transfected Prolactin Receptor Tumor Xenografts.

Antibodies labeled with positron-emitting isotopes have been used for tumor detection, predicting which patients may respond to tumor antigen-directed therapy, and assessing pharmacodynamic effects of drug interventions. Prolactin receptor (PRLR) is overexpressed in breast and prostate cancers and is a new target for cancer therapy. We evaluated REGN2878, an anti-PRLR monoclonal antibody, as an immunoPET reagent. REGN2878 was labeled with Zr-89 after conjugation with desferrioxamine B or labeled with I-131/I-124. In vitro determination of the half-maximal inhibitory concentration (IC50) of parental REGN2878, DFO-REGN2878, and iodinated REGN2878 was performed by examining the effect of the increasing amounts of these on uptake of trace-labeled I-131 REGN2878. REGN1932, a non-PRLR binding antibody, was used as a control. Imaging and biodistribution studies were performed in mice bearing tumor xenografts with various expression levels of PRLR, including MCF-7, transfected MCF-7/PRLR, PC3, and transfected PC3/PRLR and T4D7v11 cell lines. The specificity of uptake in tumors was evaluated by comparing Zr-89 REGN2878 and REGN1932, and in vivo competition compared Zr-89 REGN2878 uptake in tumor xenografts with and without prior injection of 2 mg of nonradioactive REGN2878. The competition binding assay of DFO-REGN2878 at ratios of 3.53-5.77 DFO per antibody showed IC50 values of 0.4917 and 0.7136 nM, respectively, compared to 0.3455 nM for parental REGN2878 and 0.3343 nM for I-124 REGN2878. Imaging and biodistribution studies showed excellent targeting of Zr-89 REGN2878 in PRLR-positive xenografts at delayed times of 189 h (presented as mean ± 1 SD, percent injected activity per mL (%IA/mL) 74.6 ± 33.8%IA/mL). In contrast, MCF-7/PRLR tumor xenografts showed a low uptake (7.0 ± 2.3%IA/mL) of control Zr-89 REGN1932 and a very low uptake and rapid clearance of I-124 REGN2878 (1.4 ± 0.6%IA/mL). Zr-89 REGN2878 has excellent antigen-specific targeting in various PRLR tumor xenograft models. We estimated, using image-based kinetic modeling, that PRLR antigen has a very rapid in vivo turnover half-life of ∼14 min from the cell membrane. Despite relatively modest estimated tumor PRLR expression numbers, PRLR-expressing cells have shown final retention of the Zr-89 REGN2878 antibody, with an uptake that appeared to be related to PRLR expression. This reagent has the potential to be used in clinical trials targeting PRLR.

Humanized Affinity-matured Monoclonal Antibody 8H9 Has Potent Antitumor Activity and Binds to FG Loop of Tumor Antigen B7-H3.

B7-H3 (CD276) is both an inhibitory ligand for natural killer cells and T cells and a tumor antigen that is widely expressed among human solid tumors. Anti-B7-H3 mouse monoclonal antibody 8H9 has been successfully used for radioimmunotherapy for patients with B7-H3(+) tumors. We present the humanization, affinity maturation, and epitope mapping of 8H9 based on structure determination, modeling, and yeast display methods. The crystal structure of ch8H9 Fab fragment was solved to 2.5-Å resolution and used as a template for humanization. By displaying the humanized 8H9 single chain Fv (scFv) on the surface of yeast, the affinity was matured by sequential random mutagenesis and fluorescence-activated cell sorting. Six mutations (three in the complementarity-determining region and three in the framework regions) were identified and incorporated into an affinity-matured humanized 8H9 construct (hu8H9-6m) and an affinity-matured chimeric 8H9 construct (ch8H9-6m). The hu8H9-6m scFv had a 160-fold improvement in affinity (0.9 nm KD) compared with parental hu8H9 scFv (144 nm KD). The IgG formats of ch8H9-6m and hu8H9-6m (nanomolar to subnanomolar KD) had 2-9-fold enhancements in affinity compared with their parental forms, potent in vitro antibody-dependent cell-mediated cytotoxicity (0.1-0.3 µg/ml EC50), and high tumor uptake in mouse xenografts. Based on in silico docking studies and experimental validation, the molecular epitope of 8H9 was determined to be dependent on the FG loop of B7-H3, a region critical to its function in immunologic blockade and unique among anti-B7-H3 antibodies published to date.

Theranostic pretargeted radioimmunotherapy of colorectal cancer xenografts in mice using picomolar affinity 86Y- or ¹77Lu-DOTA-Bn binding scFv C825/GPA33 IgG bispecific immunoconjugates.

PURPOSE: GPA33 is a colorectal cancer (CRC) antigen with unique retention properties after huA33-mediated tumor targeting. We tested a pretargeted radioimmunotherapy (PRIT) approach for CRC using a tetravalent bispecific antibody with dual specificity for GPA33 tumor antigen and DOTA-Bn-(radiolanthanide metal) complex. METHODS: PRIT was optimized in vivo by titrating sequential intravenous doses of huA33-C825, the dextran-based clearing agent, and the C825 haptens (177)Lu-or (86)Y-DOTA-Bn in mice bearing the SW1222 subcutaneous (s.c.) CRC xenograft model. RESULTS: Using optimized PRIT, therapeutic indices (TIs) for tumor radiation-absorbed dose of 73 (tumor/blood) and 12 (tumor/kidney) were achieved. Estimated absorbed doses (cGy/MBq) to tumor, blood, liver, spleen, and kidney for single-cycle PRIT were 65.8, 0.9 (TI 73), 6.3 (TI 10), 6.6 (TI 10), and 5.3 (TI 12), respectively. Two cycles of PRIT (66.6 or 111 MBq (177)Lu-DOTA-Bn) were safe and effective, with a complete response of established s.c. tumors (100 - 700 mm(3)) in nine of nine mice, with two mice alive without recurrence at >140 days. Tumor log kill in this model was estimated to be 2.1 - 3.0 based on time to 500-mm(3) tumor recurrence. In addition, PRIT dosimetry/diagnosis was performed by PET imaging of the positron-emitting DOTA hapten (86)Y-DOTA-Bn. CONCLUSION: We have developed anti-GPA33 PRIT as a triple-step theranostic strategy for preclinical detection, dosimetry, and safe targeted radiotherapy of established human colorectal mouse xenografts.

Pairwise comparison of 89Zr- and 124I-labeled cG250 based on positron emission tomography imaging and nonlinear immunokinetic modeling: in vivo carbonic anhydrase IX receptor binding and internalization in mouse xenografts of clear-cell renal cell carcinoma.

PURPOSE: The PET tracer, (124)I-cG250, directed against carbonic anhydrase IX (CAIX) shows promise for presurgical diagnosis of clear-cell renal cell carcinoma (ccRCC) (Divgi et al. in Lancet Oncol 8:304-310, 2007; Divgi et al. in J Clin Oncol 31:187-194, 2013). The radiometal (89)Zr, however, may offer advantages as a surrogate PET nuclide over (124)I in terms of greater tumor uptake and retention (Rice et al. in Semin Nucl Med 41:265-282, 2011). We have developed a nonlinear immunokinetic model to facilitate a quantitative comparison of absolute uptake and antibody turnover between (124)I-cG250 and (89)Zr-cG250 using a human ccRCC xenograft tumor model in mice. We believe that this unique model better relates quantitative imaging data to the salient biological features of tumor antibody-antigen binding and turnover. METHODS: We conducted experiments with (89)Zr-cG250 and (124)I-cG250 using a human ccRCC cell line (SK-RC-38) to characterize the binding affinity and internalization kinetics of the two tracers in vitro. Serial PET imaging was performed in mice bearing subcutaneous ccRCC tumors to simultaneously detect and quantify time-dependent tumor uptake in vivo. Using the known specific activities of the two tracers, the equilibrium rates of antibody internalization and turnover in the tumors were derived from the PET images using nonlinear compartmental modeling. RESULTS: The two tracers demonstrated virtually identical tumor cell binding and internalization but showed markedly different retentions in vitro. Superior PET images were obtained using (89)Zr-cG250, owing to the more prolonged trapping of the radiolabel in the tumor and simultaneous washout from normal tissues. Estimates of cG250/CAIX complex turnover were 1.35 - 5.51 × 10(12) molecules per hour per gram of tumor (20 % of receptors internalized per hour), and the ratio of (124)I/(89)Zr atoms released per unit time by tumor was 17.5. CONCLUSION: Pairwise evaluation of (89)Zr-cG250 and (124)I-cG250 provided the basis for a nonlinear immunokinetic model which yielded quantitative information about the binding and internalization of radioantibody bound to CAIX on tumor cells in vivo. (89)Zr-cG250 is likely to provide high-quality PET images and may be a useful tool to quantify CAIX/cG250 receptor turnover and cG250-accessible antigen density noninvasively in humans.

89Zr-huJ591 immuno-PET imaging in patients with advanced metastatic prostate cancer.

PURPOSE: Given the bone tropism of prostate cancer, conventional imaging modalities poorly identify or quantify metastatic disease. (89)Zr-huJ591 positron emission tomography (PET) imaging was performed in patients with metastatic prostate cancer to analyze and validate this as an imaging biomarker for metastatic disease. The purpose of this initial study was to assess safety, biodistribution, normal organ dosimetry, and optimal imaging time post-injection for lesion detection. METHODS: Ten patients with metastatic prostate cancer received 5 mCi of (89)Zr-huJ591. Four whole-body scans with multiple whole-body count rate measurements and serum activity concentration measurements were obtained in all patients. Biodistribution, clearance, and lesion uptake by (89)Zr-huJ591 immuno-PET imaging was analyzed and dosimetry was estimated using MIRD techniques. Initial assessment of lesion targeting of (89)Zr-huJ591 was done. Optimal time for imaging post-injection was determined. RESULTS: The dose was well tolerated with mild chills and rigors seen in two patients. The clearance of (89)Zr-huJ591 from serum was bi-exponential with biological half-lives of 7 ± 4.5 h (range 1.1-14 h) and 62 ± 13 h (range 51-89 h) for initial rapid and later slow phase. Whole-body biological clearance was 219 ± 48 h (range 153-317 h). The mean whole-body and liver residence time was 78.7 and 25.6 h, respectively. Dosimetric estimates to critical organs included liver 7.7 ± 1.5 cGy/mCi, renal cortex 3.5 ± 0.4 cGy/mCi, and bone marrow 1.2 ± 0.2 cGy/mCi. Optimal time for patient imaging after injection was 7 ± 1 days. Lesion targeting of bone or soft tissue was seen in all patients. Biopsies were performed in 8 patients for a total 12 lesions, all of which were histologically confirmed as metastatic prostate cancer. One biopsy-proven lesion was not positive on (89)Zr-huJ591, while the remaining 11 lesions were (89)Zr-huJ591 positive. Two biopsy-positive nodal lesions were noted only on (89)Zr-huJ591 study, while the conventional imaging modality was negative. CONCLUSION: (89)Zr-huJ591 PET imaging of prostate-specific membrane antigen expression is safe and shows good localization of disease in prostate cancer patients. Liver is the critical organ for dosimetry, and 7 ± 1 days is the optimal imaging time. A larger study is underway to determine lesion detection in an expanded cohort of patients with metastatic prostate cancer.

Evaluation of glycodendron and synthetically modified dextran clearing agents for multistep targeting of radioisotopes for molecular imaging and radioimmunotherapy.

A series of N-acetylgalactosamine-dendrons (NAG-dendrons) and dextrans bearing biotin moieties were compared for their ability to complex with and sequester circulating bispecific antitumor antibody streptavidin fusion protein (scFv4-SA) in vivo, to improve tumor-to-normal tissue concentration ratios for multistep targeted (MST) radioimmunotherapy and diagnosis. Specifically, a total of five NAG-dendrons employing a common synthetic scaffold structure containing 4, 8, 16, or 32 carbohydrate residues and a single biotin moiety were prepared (NAGB), and for comparative purposes, a biotinylated-dextran with an average molecular weight of 500 kD was synthesized from amino-dextran (DEXB). One of the NAGB compounds, CA16, has been investigated in humans; our aim was to determine if other NAGB analogues (e.g., CA8 or CA4) were bioequivalent to CA16 and/or better suited as MST reagents. In vivo studies included dynamic positron-emission tomography (PET) imaging of (124)I-labeled-scFv4-SA clearance and dual-label biodistribution studies following MST directed at subcutaneous (s.c.) human colon adenocarcinoma xenografts in mice. The MST protocol consists of three injections: first, a scFv4-SA specific for an antitumor-associated glycoprotein (TAG-72); second, CA16 or other clearing agent; and third, radiolabeled biotin. We observed using PET imaging of the (124)I-labeled-scFv4-SA clearance that the spatial arrangement of ligands conjugated to NAG (i.e., biotin linked with an extended spacer, referred to herein as long-chain (LC)) can impact the binding to the antibody in circulation and subsequent liver uptake of the NAG-antibody complex. Also, NAGB CA32-LC or CA16-LC can be utilized during MST to achieve comparable tumor-to-blood ratios and absolute tumor uptake seen previously with CA16. Finally, DEXB was equally effective as NAGB CA32-LC at lowering scFv4-SA in circulation, but at the expense of reducing absolute tumor uptake of radiolabeled biotin.

N-acetylgalactosamino dendrons as clearing agents to enhance liver targeting of model antibody-fusion protein.

Dendrimer clearing agents represent a unique class of compounds for use in multistep targeting (MST) in radioimmunotherapy and imaging. These compounds were developed to facilitate the removal of excess tumor-targeting monoclonal antibody (mAb) prior to administration of the radionuclide to minimize exposure of normal tissue to radiation. Clearing agents are designed to capture the circulating mAb, and target it to the liver for metabolism. Glycodendrons are ideally suited for MST applications as these highly branched compounds are chemically well-defined, thus advantageous over heterogeneous macromolecules. Previous studies have described glycodendron 3 as a clearing agent for use in three-step MST protocols, and early in vivo assessment of 3 showed promise. However, synthetic challenges have hampered its availability for further development. In this report we describe a new sequence of chemical steps which enables the straightforward synthesis and analytical characterization of this class of dendrons. With accessibility and analytical identification solved, we sought to evaluate both lower and higher generation dendrons for hepatocyte targeting as well as clearance of a model protein. We prepared a series of clearing agents where a single biotin is connected to glycodendrons displaying four, eight, sixteen or thirty-two α-thio-N-acetylgalactosamine (α-SGalNAc) units, resulting in compounds with molecular weights ranging from 2 to 17 kDa, respectively. These compounds were fully characterized by LCMS and NMR. We then evaluated the capacity of these agents to clear a model (131)I-labeled single chain variable fragment antibody-streptavidin ((131)I-scFv-SAv) fusion protein from blood and tissue in mice, and compared their clearing efficiencies to that of a 500 kDa dextran-biotin conjugate. Glycodendrons and dextran-biotin exhibited enhanced blood clearance of the scFv-SAv construct. Biodistribution analysis showed liver targeting/uptake of the scFv-SAv construct to be 2-fold higher for compounds 1 to 4, as well as for the 500 kDa dextran, over saline. Additionally, the data suggest the glycodendrons clear through the liver, whereas the dextran through reticuloendothelial system (RES) metabolism.

(18)F-labeled-bioorthogonal liposomes for in vivo targeting.

Liposomes are attractive vehicles for the controlled release of drugs and cytotoxins and have a long-standing history in medical research and clinical practice. In addition to established therapeutic indications, liposomes have several favorable properties for molecular imaging, including high stability and the ability to be labeled with radioisotopes, as well as paramagnetic and fluorescent contrast agents. However, long circulation times and difficulties in creating targeted liposomes have proven challenges for imaging. In this study, we have addressed these limitations using a recently developed strategy for bioorthogonal conjugation, the reaction between tetrazines and trans-cyclooctenes. By coating radiolabeled liposomes with trans-cyclooctene and pretargeting with a tetrazine coupled to a targeted peptide, we were able to selectively enhance the retention of liposomes and bind them to tumor tissue in live animals. The rapid reaction between tetrazines and trans-cyclooctenes allowed imaging to be performed with the short-lived PET tracer (18)F, yielding signal-to-background activity ratios of 7:1. The covalent, bioorthogonally driven tumor-targeting of liposomes by in vivo click chemistry is promising and should be explored for more selective and rapid delivery of radiodiagnostics and radiotherapeutics, two classes of drugs which particularly benefit from fast clearance, low nonspecific binding, and the associated reduced toxicity to kidneys and bone marrow.

Efficacy of HER2-Targeted Intraperitoneal 225Ac α-Pretargeted Radioimmunotherapy for Small-Volume Ovarian Peritoneal Carcinomatosis.

Epithelial ovarian cancer (EOC) is often asymptomatic and presents clinically in an advanced stage as widespread peritoneal microscopic disease that is generally considered to be surgically incurable. Targeted α-therapy with the α-particle-emitting radionuclide 225Ac (half-life, 9.92 d) is a high-linear-energy-transfer treatment approach effective for small-volume disease and even single cells. Here, we report the use of human epidermal growth factor receptor 2 (HER2) 225Ac-pretargeted radioimmunotherapy (PRIT) to treat a mouse model of human EOC SKOV3 xenografts growing as peritoneal carcinomatosis (PC). Methods: On day 0, 105 SKOV3 cells transduced with a luciferase reporter gene were implanted intraperitoneally in nude mice, and tumor engraftment was verified by bioluminescent imaging (BLI). On day 15, treatment was started using 1 or 2 cycles of 3-step anti-HER2 225Ac-PRIT (37 kBq/cycle as 225Ac-Proteus DOTA), separated by a 1-wk interval. Efficacy and toxicity were monitored for up to 154 d. Results: Untreated PC-tumor-bearing nude mice showed a median survival of 112 d. We used 2 independent measures of response to evaluate the efficacy of 225Ac-PRIT. First, a greater proportion of the treated mice (9/10 1-cycle and 8/10 2-cycle; total, 17/20; 85%) survived long-term compared with controls (9/27, 33%), and significantly prolonged survival was documented (log-rank [Mantel-Cox] P = 0.0042). Second, using BLI, a significant difference in the integrated BLI signal area to 98 d was noted between controls and treated groups (P = 0.0354). Of a total of 8 mice from the 2-cycle treatment group (74 kBq total) that were evaluated by necropsy, kidney radiotoxicity was mild and did not manifest itself clinically (normal serum blood urea nitrogen and creatinine). Dosimetry estimates (relative biological effectiveness-weighted dose, where relative biological effectiveness = 5) per 37 kBq administered for tumors and kidneys were 56.9 and 16.1 Gy, respectively. One-cycle and 2-cycle treatments were equally effective. With immunohistology, mild tubular changes attributable to α-toxicity were observed in both therapeutic groups. Conclusion: Treatment of EOC PC-tumor-bearing mice with anti-HER2 225Ac-PRIT resulted in histologic cures and prolonged survival with minimal toxicity. Targeted α-therapy using the anti-HER2 225Ac-PRIT system is a potential treatment for otherwise incurable EOC.

Engineering CAR-T cells for radiohapten capture in imaging and radioimmunotherapy applications.

Rationale: The in vivo dynamics of CAR-T cells remain incompletely understood. Novel methods are urgently needed to longitudinally monitor transferred cells non-invasively for biodistribution, functionality, proliferation, and persistence in vivo and for improving their cytotoxic potency in case of treatment failure. Methods: Here we engineered CD19 CAR-T cells ("Thor"-cells) to express a membrane-bound scFv, huC825, that binds DOTA-haptens with picomolar affinity suitable for labeling with imaging or therapeutic radionuclides. We assess its versatile utility for serial tracking studies with PET and delivery of α-radionuclides to enhance anti-tumor killing efficacy in sub-optimal adoptive cell transfer in vivo using Thor-cells in lymphoma models. Results: We show that this reporter gene/probe platform enables repeated, sensitive, and specific assessment of the infused Thor-cells in the whole-body using PET/CT imaging with exceptionally high contrast. The uptake on PET correlates with the Thor-cells on a cellular and functional level. Furthermore, we report the ability of Thor-cells to accumulate cytotoxic alpha-emitting radionuclides preferentially at tumor sites, thus increasing therapeutic potency. Conclusion: Thor-cells are a new theranostic agent that may provide crucial information for better and safer clinical protocols of adoptive T cell therapies, as well as accelerated development strategies.

Pretargeting: A Path Forward for Radioimmunotherapy.

Pretargeted radioimmunodiagnosis and radioimmunotherapy aim to efficiently combine antitumor antibodies and medicinal radioisotopes for high-contrast imaging and high-therapeutic-index (TI) tumor targeting, respectively. As opposed to conventional radioimmunoconjugates, pretargeted approaches separate the tumor-targeting step from the payload step, thereby amplifying tumor uptake while reducing normal-tissue exposure. Alongside contrast and TI, critical parameters include antibody immunogenicity and specificity, availability of radioisotopes, and ease of use in the clinic. Each of the steps can be optimized separately; as modular systems, they can find broad applications irrespective of tumor target, tumor type, or radioisotopes. Although this versatility presents enormous opportunity, pretargeting is complex and presents unique challenges for clinical translation and optimal use in patients. The purpose of this article is to provide a brief historical perspective on the origins and development of pretargeting strategies in nuclear medicine, emphasizing 2 protein delivery systems that have been extensively evaluated (i.e., biotin-streptavidin and hapten-bispecific monoclonal antibodies), as well as radiohaptens and radioisotopes. We also highlight recent innovations, including pretargeting with bioorthogonal chemistry and novel protein vectors (such as self-assembling and disassembling proteins and Affibody molecules). We caution the reader that this is by no means a comprehensive review of the past 3 decades of pretargeted radioimmunodiagnosis and pretargeted radioimmunotherapy. But we do aim to highlight major developmental milestones and to identify benchmarks for success with regard to TI and toxicity in preclinical models and clinically. We believe this approach will lead to the identification of key obstacles to clinical success, revive interest in the utility of radiotheranostics applications, and guide development of the next generation of pretargeted theranostics.

Intraperitoneal Pretargeted Radioimmunotherapy for Colorectal Peritoneal Carcinomatosis.

Peritoneal carcinomatosis (PC) is considered incurable, and more effective therapies are needed. Herein we test the hypothesis that GPA33-directed intracompartmental pretargeted radioimmunotherapy (PRIT) can cure colorectal peritoneal carcinomatosis. Nude mice were implanted intraperitoneally with luciferase-transduced GPA33-expressing SW1222 cells for aggressive peritoneal carcinomatosis (e.g., resected tumor mass 0.369 ± 0.246 g; n = 17 on day 29). For GPA33-PRIT, we administered intraperitoneally a high-affinity anti-GPA33/anti-DOTA bispecific antibody (BsAb), followed by clearing agent (intravenous), and lutetium-177 (Lu-177) or yttrium-86 (Y-86) radiolabeled DOTA-radiohapten (intraperitoneal) for beta/gamma-emitter therapy and PET imaging, respectively. The DOTA-radiohaptens were prepared from S-2-(4-aminobenzyl)-1,4,7, 10-tetraazacyclododecane tetraacetic acid chelate (DOTA-Bn). Efficacy and toxicity of single- versus three-cycle therapy were evaluated in mice 26-27 days post-tumor implantation. Single-cycle treatment ([177Lu]LuDOTA-Bn 111 MBq; tumor dose: 4,992 cGy) significantly prolonged median survival (MS) approximately 2-fold to 84.5 days in comparison with controls (P = 0.007). With three-cycle therapy (once weekly, total 333 MBq; tumor dose: 14,975 cGy), 6/8 (75%) survived long-term (MS > 183 days). Furthermore, for these treated long-term survivors, 1 mouse was completely disease free (microscopic "cure") at necropsy; the others showed stabilized disease, which was detectable during PET-CT using [86Y]DOTA-Bn. Treatment controls had MS ranging from 42-52.5 days (P < 0.001) and 19/20 mice succumbed to progressive intraperitoneal disease by 69 days. Multi-cycle GPA33 DOTA-PRIT significantly prolongs survival with reversible myelosuppression and no chronic marrow (929 cGy to blood) or kidney (982 cGy) radiotoxicity, with therapeutic indices of 12 for blood and 12 for kidneys. MTD was not reached.

A Self-Assembling and Disassembling (SADA) Bispecific Antibody (BsAb) Platform for Curative Two-step Pretargeted Radioimmunotherapy.

PURPOSE: Many cancer treatments suffer from dose-limiting toxicities to vital organs due to poor therapeutic indices. To overcome these challenges we developed a novel multimerization platform that rapidly removes tumor-targeting proteins from the blood to substantially improve therapeutic index. EXPERIMENTAL DESIGN: The platform was designed as a fusion of a self-assembling and disassembling (SADA) domain to a tandem single-chain bispecific antibody (BsAb, anti-ganglioside GD2 × anti-DOTA). SADA-BsAbs were assessed with multiple in vivo tumor models using two-step pretargeted radioimmunotherapy (PRIT) to evaluate tumor uptake, dosimetry, and antitumor responses. RESULTS: SADA-BsAbs self-assembled into stable tetramers (220 kDa), but could also disassemble into dimers or monomers (55 kDa) that rapidly cleared via renal filtration and substantially reduced immunogenicity in mice. When used with rapidly clearing DOTA-caged PET isotopes, SADA-BsAbs demonstrated accurate tumor localization, dosimetry, and improved imaging contrast by PET/CT. When combined with therapeutic isotopes, two-step SADA-PRIT safely delivered massive doses of alpha-emitting (225Ac, 1.48 MBq/kg) or beta-emitting (177Lu, 6,660 MBq/kg) S-2-(4-aminobenzyl)-1,4,7,10-tetraazacyclododecane tetraacetic acid (DOTA) payloads to tumors, ablating them without any short-term or long-term toxicities to the bone marrow, kidneys, or liver. CONCLUSIONS: The SADA-BsAb platform safely delivered large doses of radioisotopes to tumors and demonstrated no toxicities to the bone marrow, kidneys, or liver. Because of its modularity, SADA-BsAbs can be easily adapted to most tumor antigens, tumor types, or drug delivery approaches to improve therapeutic index and maximize the delivered dose.See related commentary by Capala and Kunos, p. 377.

Alpha radioimmunotherapy using 225Ac-proteus-DOTA for solid tumors - safety at curative doses.

This is the initial report of an α-based pre-targeted radioimmunotherapy (PRIT) using 225Ac and its theranostic pair, 111In. We call our novel tumor-targeting DOTA-hapten PRIT system "proteus-DOTA" or "Pr." Herein we report the first results of radiochemistry development, radiopharmacology, and stoichiometry of tumor antigen binding, including the role of specific activity, anti-tumor efficacy, and normal tissue toxicity with the Pr-PRIT approach (as α-DOTA-PRIT). A series of α-DOTA-PRIT therapy studies were performed in three solid human cancer xenograft models of colorectal cancer (GPA33), breast cancer (HER2), and neuroblastoma (GD2), including evaluation of chronic toxicity at ~20 weeks of select survivors. Methods: Preliminary biodistribution experiments in SW1222 tumor-bearing mice revealed that 225Ac could not be efficiently pretargeted with current DOTA-Bn hapten utilized for 177Lu or 90Y, leading to poor tumor uptake in vivo. Therefore, we synthesized Pr consisting of an empty DOTA-chelate for 225Ac, tethered via a short polyethylene glycol linker to a lutetium-complexed DOTA for picomolar anti-DOTA chelate single-chain variable fragment (scFv) binding. Pr was radiolabeled with 225Ac and its imaging surrogate, 111In. In vitro studies verified anti-DOTA scFv recognition of [225Ac]Pr, and in vivo biodistribution and clearance studies were performed to evaluate hapten suitability and in vivo targeting efficiency. Results: Intravenously (i.v.) administered 225Ac- or 111In-radiolabeled Pr in mice showed rapid renal clearance and minimal normal tissue retention. In vivo pretargeting studies show high tumor accumulation of Pr (16.71 ± 5.11 %IA/g or 13.19 ± 3.88 %IA/g at 24 h p.i. for [225Ac]Pr and [111In]Pr, respectively) and relatively low uptake in normal tissues (all average ≤ 1.4 %IA/g at 24 h p.i.). Maximum tolerated dose (MTD) was not reached for either [225Ac]Pr alone or pretargeted [225Ac]Pr at administered activities up to 296 kBq/mouse. Single-cycle treatment consisting of α-DOTA-PRIT with either huA33-C825 bispecific anti-tumor/anti-DOTA-hapten antibody (BsAb), anti-HER2-C825 BsAb, or hu3F8-C825 BsAb for targeting GPA33, HER2, or GD2, respectively, was highly effective. In the GPA33 model, no complete responses (CRs) were observed but prolonged overall survival of treated animals was 42 d for α-DOTA-PRIT vs. 25 d for [225Ac]Pr only (P < 0.0001); for GD2, CRs (7/7, 100%) and histologic cures (4/7, 57%); and for HER2, CRs (7/19, 37%) and histologic cures (10/19, 56%) with no acute or chronic toxicity. Conclusions: [225Ac]Pr and its imaging biomarker [111In]Pr demonstrate optimal radiopharmacologic behavior for theranostic applications of α-DOTA-PRIT. For this initial evaluation of efficacy and toxicity, single-cycle treatment regimens were performed in all three systems. Histologic toxicity was not observed, so MTD was not observed. Prolonged overall survival, CRs, and histologic cures were observed in treated animals. In comparison to RIT with anti-tumor IgG antibodies, [225Ac]Pr has a much improved safety profile. Ultimately, these data will be used to guide clinical development of toxicity and efficacy studies of [225Ac]Pr, with the goal of delivering massive lethal doses of radiation to achieve a high probability of cure without toxicity.

Mapping protein-protein interactions by localized oxidation: consequences of the reach of hydroxyl radical.

Hydroxyl radicals generated from a variety of methods, including not only synchrotron radiation but also Fenton reactions involving chelated iron, have become an accepted macromolecular footprinting tool. Hydroxyl radicals react with proteins via multiple mechanisms that lead to both polypeptide backbone cleavage events and side chain modifications (e.g., hydroxylation and carbonyl formation). The use of site-specifically tethered iron chelates can reveal protein-protein interactions, but the interpretation of such experiments will be strengthened by improving our understanding of how hydroxyl radicals produced at a point on a protein react with other protein sites. We have developed methods for monitoring carbonyl formation on proteins as a function of distance from a hydroxyl generator, iron-(S)-1-[p-(bromoacetamido)benzyl]EDTA (FeBABE), conjugated to an engineered cysteine residue. After activation of the chelated iron with ascorbate and peroxide produces new protein carbonyl groups, their positions can be identified using element-coded affinity tagging (ECAT), with carbonyl-specific tags {e.g., rare earth chelates of (S)-2-[4-(2-aminooxy)acetamidobenzyl]-1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid (AOD)} that allow for affinity purification, identification, and relative quantitation of oxidation sites using mass spectrometry. Intraprotein oxidation of single-cysteine mutants of Escherichia coli sigma(70) by tethered FeBABE was used to calibrate the reach of hydroxyl radical by comparison to the crystal structure; the application to protein-protein interactions was demonstrated using the same sigma(70) FeBABE conjugates in complexes with the RNA polymerase core enzyme. The results provide fundamental information for interpreting protein footprinting experiments in other systems.

Imaging Patients with Metastatic Castration-Resistant Prostate Cancer Using 89Zr-DFO-MSTP2109A Anti-STEAP1 Antibody.

Six-transmembrane epithelial antigen of prostate-1 (STEAP1) is a relatively newly identified target in prostate cancer. We evaluated the ability of PET/CT with 89Zr-DFO-MSTP2109A, an antibody that recognizes STEAP1, to detect lesions in patients with metastatic castration-resistant prostate cancer (mCRPC). Methods: Nineteen mCRPC patients were prospectively imaged using approximately 185 MBq/10 mg of 89Zr-DFO-MSTP2109A. 89Zr-DFO-MSTP2109A PET/CT images obtained 4-7 d after injection were compared with bone and CT scans. Uptake in lesions was measured. Fifteen patients were treated with an antibody-drug conjugate (ADC) based on MSTP2109A; ADC treatment-related data were correlated with tumor uptake by PET imaging. Bone or soft-tissue biopsy samples were evaluated. Results: No significant toxicity occurred. Excellent uptake was observed in bone and soft-tissue disease. Median SUVmax was 20.6 in bone and 16.8 in soft tissue. Sixteen of 17 lesions biopsied were positive on 89Zr-DFO-MSTP2109A, and all sites were histologically positive (1 on repeat biopsy). Bayesian analysis resulted in a best estimate of 86% of histologically positive lesions being true-positive on imaging (95% confidence interval, 75%-100%). There was no correlation between SUVmax tumor uptake and STEAP1 immunohistochemistry, survival after ADC treatment, number of ADC treatment cycles, or change in prostate-specific antigen level. Conclusion:89Zr-DFO-MSTP2109A is well tolerated and shows localization in mCRPC sites in bone and soft tissue. Given the high SUV in tumor and localization of a large number of lesions, this reagent warrants further exploration as a companion diagnostic in patients undergoing STEAP1-directed therapy.