immuno-PET Studies Research Articles

Facile labelling of an anti-epidermal growth factor receptor Nanobody with 68Ga via a novel bifunctional desferal chelate for immuno-PET

PurposeThe ∼15 kDa variable domains of camelid heavy-chain-only antibodies (called Nanobodies®) have the flexibility to be formatted as monovalent, monospecific, multivalent or multispecific single chain proteins with either fast or slow pharmacokinetics. We report the evaluation of the fast kinetic anti-epidermal growth factor receptor (EGFR) Nanobody 7D12, labelled with 68Ga via the novel bifunctional chelate (BFC) p-isothiocyanatobenzyl-desferrioxamine (Df-Bz-NCS). Df-Bz-NCS has recently been introduced as the chelate of choice for 89Zr immuno-positron emission tomography (PET).MethodsNanobody 7D12 was premodified with Df-Bz-NCS at pH 9. Radiolabelling with purified 68Ga was performed at pH 5.0–6.5 for 5 min at room temperature. For in vitro stability measurements in storage buffer (0.25 M NaOAc with 5 mg ml−1 gentisic acid, pH 5.5) at 4°C or in human serum at 37°C, a mixture of 67Ga and 68Ga was used. Biodistribution and immuno-PET studies of 68Ga-Df-Bz-NCS-7D12 were performed in nude mice bearing A431 xenografts using 89Zr-Df-Bz-NCS-7D12 as the reference conjugate.ResultsThe Df-Bz-NCS chelate was conjugated to Nanobody 7D12 with a chelate to Nanobody molar substitution ratio of 0.2:1. The overall 68Ga radiochemical yield was 55–70% (not corrected for decay); specific activity was 100–500 MBq/mg. Radiochemical purity of the conjugate was >96%, while the integrity and immunoreactivity were preserved. 68/67Ga-Df-Bz-NCS-7D12 was stable in storage buffer as well as in human serum during a 5-h incubation period (<2% radioactivity loss). In biodistribution studies the 68Ga-labelled Nanobody 7D12 showed high uptake in A431 tumours (ranging from 6.1 ± 1.3 to 7.2 ± 1.5%ID/g at 1–3 h after injection) and high tumour to blood ratios, which increased from 8.2 to 14.4 and 25.7 at 1, 2 and 3 h after injection, respectively. High uptake was also observed in the kidneys. Biodistribution was similar to that of the reference conjugate 89Zr-Df-Bz-NCS-7D12. Tumours were clearly visualized in a PET imaging study.ConclusionVia a rapid procedure under mild conditions a 68Ga-Nanobody was obtained that exhibited high tumour uptake and tumour to normal tissue ratios in nude mice bearing A431 xenografts. Fast kinetic 68Ga-Nanobody conjugates can be promising tools for tumour detection and imaging of target expression.

Immuno-Positron Emission Tomography: Shedding Light on Clinical Antibody Therapy

Summation Monoclonal antibodies (mAbs) have been approved for therapeutic use in a broad range of medical indications, especially in oncology, and are forming the most rapidly expanding category of pharmaceuticals. Although engineered mAb fragments and nontraditional antibody-like scaffolds are receiving increasingly more attention, most of the mAb candidates evaluated in past and ongoing clinical trials are full-length mAbs. Immuno-positron emission tomography (PET), the tracking and quantification of mAbs with PET in vivo at superior imaging quality, is an exciting novel option for better understanding the in vivo behavior and efficacy of mAbs in individual patients. This review focuses on immuno-PET with full-length mAbs, and the associated use of the long-lived positron emitters zirconium-89 ((89)Zr) and iodine-124 ((124)I). Very recently, crucial achievements have been obtained to allow broad-scale application of (89)Zr- and (124)I-immuno-PET in clinical mAb development and applications. (89)Zr and (124)I became commercially available worldwide for clinical use. A chelate for facile coupling of (89)Zr to mAbs became commercially available, and generic procedures for labeling of mAbs with (89)Zr and (124)I in a current good manufacturing practice compliant way were established. In this review, critical aspects for the translation of immuno-PET from preclinical investigations to clinical trials will be discussed, as well as the potential clinical applications of immuno-PET. An overview of the results of the first clinical immuno-PET studies will be provided.

89Zr-DFO-J591 for ImmunoPET of Prostate-Specific Membrane Antigen Expression In Vivo

(89)Zr (half-life, 78.41 h) is a positron-emitting radionuclide that displays excellent potential for use in the design and synthesis of radioimmunoconjugates for immunoPET. In the current study, we report the preparation of (89)Zr-desferrioxamine B (DFO)-J591, a novel (89)Zr-labeled monoclonal antibody (mAb) construct for targeted immunoPET and quantification of prostate-specific membrane antigen (PSMA) expression in vivo. The in vivo behavior of (89)Zr-chloride, (89)Zr-oxalate, and (89)Zr-DFO was studied using PET. High-level computational studies using density functional theory calculations have been used to investigate the electronic structure of (89)Zr-DFO and probe the nature of the complex in aqueous conditions. (89)Zr-DFO-J591 was characterized both in vitro and in vivo. ImmunoPET in male athymic nu/nu mice bearing subcutaneous LNCaP (PSMA-positive) or PC-3 (PSMA-negative) tumors was conducted. The change in (89)Zr-DFO-J591 tissue uptake in response to high- and low-specific-activity formulations in the 2 tumor models was measured using acute biodistribution studies and immunoPET. The basic characterization of 3 important reagents-(89)Zr-chloride, (89)Zr-oxalate, and the complex (89)Zr-DFO-demonstrated that the nature of the (89)Zr species dramatically affects the biodistribution and pharmacokinetics. Density functional theory calculations provide a rationale for the observed high in vivo stability of (89)Zr-DFO-labeled mAbs and suggest that in aqueous conditions, (89)Zr-DFO forms a thermodynamically stable, 8-coordinate complex by coordination of 2 water molecules. (89)Zr-DFO-J591 was produced in high radiochemical yield (>77%) and purity (>99%), with a specific activity of 181.7 +/- 1.1 MBq/mg (4.91 +/- 0.03 mCi/mg). In vitro assays demonstrated that (89)Zr-DFO-J591 had an initial immunoreactive fraction of 0.95 +/- 0.03 and remained active for up to 7 d. In vivo biodistribution experiments revealed high, target-specific uptake of (89)Zr-DFO-J591 in LNCaP tumors after 24, 48, 96, and 144 h (34.4 +/- 3.2 percentage injected dose per gram [%ID/g], 38.0 +/- 6.2 %ID/g, 40.4 +/- 4.8 %ID/g, and 45.8 +/- 3.2 %ID/g, respectively). ImmunoPET studies also showed that (89)Zr-DFO-J591 provides excellent image contrast, with tumor-to-muscle ratios greater than 20, for the delineation of LNCaP xenografts between 48 and 144 h after administration. These studies demonstrate that (89)Zr-DFO-labeled mAbs show exceptional promise as radiotracers for immunoPET of human cancers. (89)Zr-DFO-J591 displays high tumor-to-background tissue contrast in immunoPET and can be used to delineate and quantify PSMA-positive prostate tumors in vivo.

Cerenkov Luminescence Imaging of Medical Isotopes

The development of novel multimodality imaging agents and techniques represents the current frontier of research in the field of medical imaging science. However, the combination of nuclear tomography with optical techniques has yet to be established. Here, we report the use of the inherent optical emissions from the decay of radiopharmaceuticals for Cerenkov luminescence imaging (CLI) of tumors in vivo and correlate the results with those obtained from concordant immuno-PET studies. In vitro phantom studies were used to validate the visible light emission observed from a range of radionuclides including the positron emitters (18)F, (64)Cu, (89)Zr, and (124)I; beta-emitter (131)I; and alpha-particle emitter (225)Ac for potential use in CLI. The novel radiolabeled monoclonal antibody (89)Zr-desferrioxamine B [DFO]-J591 for immuno-PET of prostate-specific membrane antigen (PSMA) expression was used to coregister and correlate the CLI signal observed with the immuno-PET images and biodistribution studies. Phantom studies confirmed that Cerenkov radiation can be observed from a range of positron-, beta-, and alpha-emitting radionuclides using standard optical imaging devices. The change in light emission intensity versus time was concordant with radionuclide decay and was also found to correlate linearly with both the activity concentration and the measured PET signal (percentage injected dose per gram). In vivo studies conducted in male severe combined immune deficient mice bearing PSMA-positive, subcutaneous LNCaP tumors demonstrated that tumor-specific uptake of (89)Zr-DFO-J591 could be visualized by both immuno-PET and CLI. Optical and immuno-PET signal intensities were found to increase over time from 24 to 96 h, and biodistribution studies were found to correlate well with both imaging modalities. These studies represent the first, to our knowledge, quantitative assessment of CLI for measuring radiotracer uptake in vivo. Many radionuclides common to both nuclear tomographic imaging and radiotherapy have the potential to be used in CLI. The value of CLI lies in its ability to image radionuclides that do not emit either positrons or gamma-rays and are, thus, unsuitable for use with current nuclear imaging modalities. Optical imaging of Cerenkov radiation emission shows excellent promise as a potential new imaging modality for the rapid, high-throughput screening of radiopharmaceuticals.

Radiation Dosimetry of 89Zr-Labeled Chimeric Monoclonal Antibody U36 as Used for Immuno-PET in Head and Neck Cancer Patients

Immuno-PET is an appealing concept in the detection of tumors and planning of antibody-based therapy. For this purpose, the long-lived positron emitter (89)Zr (half-life, 78.4 h) recently became available. The aim of the present first-in-humans (89)Zr immuno-PET study was to assess safety, biodistribution, radiation dose, and quantification of the (89)Zr-labeled chimeric monoclonal antibody (cmAb) U36 in patients with head and neck squamous cell carcinoma (HNSCC). In addition, the performance of immuno-PET for detecting lymph node metastases was evaluated, as described previously. Twenty HNSCC patients, scheduled to undergo surgical tumor resection, received 75 MBq of (89)Zr-cmAb U36 (10 mg). Immuno-PET scans were acquired at 1, 24, 72, or 144 h after injection. The biodistribution of the radioimmunoconjugate was evaluated by ex vivo radioactivity measurement in blood and in biopsies from the surgical specimen obtained at 168 h after injection. Uptake levels and residence times in blood, tumors, and organs of interest were derived from quantitative immuno-PET studies, and absorbed doses were calculated using OLINDA/EXM 1.0. The red marrow dose was calculated using the residence time for blood. (89)Zr-cmAb U36 was well tolerated by all subjects. PET quantification of blood-pool activity in the left ventricle of the heart showed a good agreement with sampled blood activity (difference equals 0.2% +/- 16.9% [mean +/- SD]) except for heavy-weight patients (>100 kg). A good agreement was also found for the assessment of mAb uptake in primary tumors (mean deviation, -8.4% +/- 34.5%). The mean absorbed red marrow dose was 0.07 +/- 0.02 mSv/MBq and 0.09 +/- 0.01 mSv/MBq in men and women, respectively. The normal organ with the highest absorbed dose was the liver (mean dose, 1.25 +/- 0.27 mSv/MBq in men and 1.35 +/- 0.21 mSv/MBq in women), thereafter followed by kidneys, thyroid, lungs, and spleen. The mean effective dose was 0.53 +/- 0.03 mSv/MBq in men and 0.66 +/- 0.03 mSv/MBq in women. Measured excretion via the urinary tract was less than 3% during the first 72 h. (89)Zr immuno-PET can be safely used to quantitatively assess biodistribution, uptake, organ residence times, and radiation dose, justifying its further clinical exploitation in the detection of tumors and planning of mAb-based therapy.