ImmunoPET Precision Imaging with 89Zr-based Antibodies
A primary area of interest of our laboratory is the design, synthesis, characterization, and validation of Zr-89-based antibodies and antibody-derived imaging agents. One of the most fundamental principles in the construction of effective antibody-based nuclear imaging agents is matching the physical half-life of the radioisotope to the pharmacokinetic half-life of the targeting vector. This exigency has led our group to exploit the positron-emitting radiometal 89Zr (t1/2 = 78.41 h) as an effective and versatile long-lived radiolabel for PET imaging. Currently, our laboratory is pursuing a wide variety of projects that employ 89Zr as a radioisotope, as shown in the following examples.
89Zr-Fab2-DFO-Gal3 for new thyroid carcinoma imaging
Thyroid nodules are very common in adults, especially in areas with iodine deficiency. Currently it is often not possible to exclude a malignant nodule by nuclear imaging (radioiodine scintigraphy), ultrasound imaging and fine needle aspiration biopsy, especially in patients with multinodular goiter, because an imaging agent with high and specific uptake in malignant lesions is lacking. A non-invasive imaging test that specifically accumulates in malignant thyroid nodules could substantially reduce the morbidity from thyroid surgeries for benign nodules and reduce healthcare costs.
Galectin-3 (gal-3) is a b-galactoside binding protein expressed in well-differentiated and in undifferentiated thyroid cancer types but not in normal thyrocytes and benign thyroid lesions. We validate gal-3 targeting as a specific method to detect radioiodine non-avid thyroid cancer in thyroid orthotopic tumor models established inoculating papillary (BcPAP) and anaplastic (CAL62 and FRO82-1) thyroid carcinoma cells lines previously characterized via WB and PCR, for gal-3 and sodium-iodide symporter (NIS) expression, in athymic nude mice. An 89Zr-labeled F(ab’)2 anti-gal-3 was generated and characterized for binding versus iodine-125 on 2D and 3D cell cultures. Head-to-head PET/CT comparison of iodine-124 versus 89Zr-DFO-F(ab’)2 anti-gal-3 was performed, followed by biodistribution studies and immunohistochemical analysis for gal-3 and NIS expression. PET/CT imaging showed 89Zr-DFO-F(ab’)2 anti-gal-3 signal associated to the orthotopic implanted tumors only, while no signal was detected in the tumor-free thyroid lobe. Conversely, PET imaging using iodine-124 showed background accumulation in tumor infiltrated lobe, a condition simulating the presence of radioiodine non-avid thyroid cancer nodules, and high accumulation in normal thyroid lobe.
Figure legend: Head to head comparison immunoPET using 89Zr-Dfo-F(ab’)2 anti-Gal-3 versus iodine-124 PET.
For the clinical translation of this new imaging approach, we have developed, in collaboration with the group of Arne Skerra (Biochemistry Depart. TUM), a humanized Fab’ anti gal-3 presenting a prolonged plasma half-life and tumor accumulation via PASylation technology.
89Zr-Fab2-DFO-Gal3 to visualize atherosclerotic plaques remodeling
Atherosclerosis is a multifactorial chronic inflammatory disorder characterized by a disturbed equilibrium of immune responses and lipid accumulation, leading to plaque development. Most atherosclerotic plaques remain clinically asymptomatic, though some undergo a series of changes that cause life-threatening complications. Visualization of atherosclerotic plaques and in particular identification of the high-risk lesions prone to rupture in vivo is a challenging objective with the ultimate goal to develop pharmacological and/or therapeutic strategies to prevent lethality. Molecular imaging offers potential opportunities to develop diagnostic approaches to assess the pathobiology of atherosclerotic plaques. Improved understanding of the molecular and biological processes has stimulated the development of imaging probes, which may aid to identify high-risk atherosclerotic lesions and to apply individually tailored interventions. Monocyte-derived macrophages are major inflammatory cells associated with atherosclerotic lesions and are recognized as key pathophysiologically important immune cells. Therefore, macrophages are gaining attention as imaging targets in atherosclerosis. Galectin-3 (Gal3) is a member of the lectin family, which is known to be involved in multiple aspects of inflammatory cell pathology, and in particular is a constitutive marker of activated macrophages. Overexpression of Gal3 in the aorta of hypercholesterolemic animals and in human atherosclerotic lesions has been reported, suggesting a direct involvement of this multifaceted protein in the pathophysiology of the disease. Gal3 has therefore been proposed as a potential target for non-invasive molecular imaging of atherosclerosis and in particular plaque vulnerability.
Figure legend: Multimodal imaging approach to confirm the expression of Galectin-3 in atherosclerotic plaques in remodeling and macrophages infiltration via 89Zr-Dfo-F(ab’)2 anti-Gal3 and fluorescent molecular tomography.
Using zirconium-89 labeled Gal3-F(ab’)2 mAb in combination with fluorescent molecular tomography imaging (in collaboration with Vasilis Ntziachristos), we have shown that nuclear imaging has the potential to visualize specific biological activities associated with plaque progression and/or instability, and that Gal3 expression in atherosclerotic plaques may be used as a novel biomarker to identify patients at high risk of cardiovascular events or to monitor the impact of treatment aimed at plaque stabilization.
89Zr-based Antibodies for Imaging T cell distribution and homing
Genetic modification of effector T cells by T-cell receptors (TCR) specifically recognizing tumor-associated peptide ligands is a highly attractive novel strategy for the treatment of diverse malignant diseases. However, in order to understand T cell trafficking, expansion and functionality in preclinical models, the development of non-invasive and highly sensitive cell-tracking technologies is necessary. Moreover, clinical translation of such technologies would further provide possibilities to improve this therapeutic approach in humans and may provide options for directed depletion.
To this purpose, in collaboration with Angela Krackhardt (III Medical Department of MRI-TUM), we have taken advantage of using an alloreactive TCR with high specificity for the leukemia associated differentiation antigen myeloperoxidase (MPO) which is used for genetic modification of central memory T-cells (TCM). To monitor TCR-transduced T cells in vivo, we used Zirconium-89 labeling of an anti-murine TCR antibody (TCRmu), which binds to the murinized part of the TCR beta chain of the introduced TCR. We could detect specific 89Zr-TCRmu binding to TCR-transduced TCM in vitro and those cells demonstrated functionality.
Figure legend: Scheme of production of TRC-transgenic T cells and tumor size-driven reactive T-cell infiltration visualized by 89Zr-Fab2 anti-TCRmu tracer.
A xenogenic mouse model was established using immunocompromized mice injected subcutaneously with the AML-cell line ML2. After tumor inoculation, we injected intravenously TCR-transduced human T cells and PET/CT imaging was performed at different time points post intravenous injection of 89Zr-labeled Fab2 fragment of the antibody which recognizes the murinised portion. In vivo application demonstrated a strong signal on the tumor site where TCR-transgenic T cells were accumulating, with a signal distribution directly correlating with the number of T cell infiltrating the tumor and with the tumor size, but no signal when untransduced T cells or PBS were used. Using this experimental approach, we have proved that a non-invasive imaging approach for tracking specifically human TCR-engineered lymphocytes in vivo is feasible. This Fab2-fragment based PET imaging modality may be useful to monitor adoptively transferred T cells transduced with diverse TCR in murine models. Moreover, this imaging modality may be suitable for clinical translation.
89Zr-based molecules as tracers for Imaging heat-shock protein 70 in tumors
The search for suitable PET tracers for tumor-specific detection in early stages and monitoring of therapy responses can improve clinical outcome. Clinically applied tracers frequently address metabolic tumor features (e.g. [18F]-FDG) or target molecules that are overexpressed in tumor cells, but are also displayed on the plasma membrane of normal cells. Consequently, these tracers might show suboptimal tumor specificity and false positive signals in non-neoplastic tissues. Therefore, novel imaging approaches focus on tracers targeting epitopes that are exclusively present on tumor cells. The membrane-bound form of the major stress inducible 72kDa heat shock protein 70 represents such a tumor-specific target. Hsp70 is expressed in the cytosol of all nucleated cells where it fulfils a variety of chaperoning functions, such as folding and assembly of nascent polypeptides, refolding of denatured proteins, as well as regulation of protein transport across membranes. The majority of human tumors of different entities is characterized by a constitutive overexpression of Hsp70 which supports tumor progression, survival, metastatic spread and resistance to therapy.
Figure legend: Representative pseudocolor PET scans of cmHsp70.1-DFO[89Zr] in a living mouse bearing sc implanted 4T1 wt tumor, 72h after iv administration (left), and of a mouse bearing sc implanted 4T1 (left shoulder) and CT26 (right neck) tumors, 24h after iv TPP-PEG24-DFO[89Zr] injection (right).
Preclinical therapeutic approaches using cmHsp70.1 mAb for targeting mHsp70 on tumor cells revealed an ample tumor-specific binding in vivo, that results in a potent activation of the hosts´ antibody dependent cellular antitumor immune response. However, due to the large size (150kDa) and the immunogenic potential, antibody-based PET probes, investigated in collaboration with Gabriele Multhoff (Radiation oncology dept. MRI-TUM), exhibit certain limitations for imaging purposes including unfavorable biodistribution kinetics caused by long blood circulation times and slow tumor uptake, high accumulation in the liver which increases the risk of hepato-radiotoxicity, and Fc receptor based off-target effects. For in vivo imaging applications, we chose to use a smaller molecule, such as the 14mer peptide TPP that mimics properties of the oligomerization domain of Hsp70 and enables a tumor-specific targeting via mHsp70. This approach shows numerous advantages over antibody including short circulation periods, fast body clearance, favorable biodistribution, improved ingress into solid tumors and highly efficient tumor cell penetration capabilities.
Development of new chelators for PET radiometal complexation
New ligands for small temperature sensitive 68Ga-radiopharmaceutical
The widespread clinical application of 68Ge-based radioisotope generators (t1=2 (68Ge)=270.8 days) for the production of the PET isotope 68Ga (t1=2=67.71 min, Eb+,max=1.89 MeV, 89% decays through positron emission), along with the favorable properties of the isotope (i. e. sufficient half-life for producing tracers that expose the patient to a minimal radiation dose), has boosted research activity aiming to design effective, specific and safe 68Ga-based radiopharmaceuticals. A 68Ga-based radiopharmaceutical consists of a thermodynamically stable and kinetically inert GaIII complex linked to a specific vector, most often represented by peptides or pseudopeptides. Ga3+ ions are typically complexed at acidic pH to avoid the formation of Ga(OH)3, by a chelator having at least 6 donor atoms (N- or O-donors) that is able to wrap around the metal ion and prevent transmetallation reactions with endogeneous metal ions (Cu2+, Zn2+, Ca2+) or ligand exchange reactions with proteins such as transferrin. An efficient 68Ga-labeling reaction, that does not require high temperatures (i. e. 958C as for DOTA-based systems) represents a very attractive opportunity when producing fragile, temperature sensitive macromolecular tracers. In this context, a polyaminopolycarboxylate heptadentate ligand based on a 1,4-diazepine scaffold (AAZTA=6-amino-6-methylperhydro-1,4-diazepinetetraacetic acid, Scheme 1) has been thoroughly studied as chelator for 68Ga, 111In and 177Lu for application in PET, SPECT and radionuclide therapy. With the aim to further improve the performance of the AAZTA chelator for labeling temperature sensitive molecules, we designed and investigated in collaboration with Lorento Tei (UNIPO, Italy) a new ligand containing a cyclohexane ring fused onto the seven-membered diazepine ring, raising its structural rigidity.
Figure legend: Cartoon showing the coordination of 68Ga from CyAAZTA chelator suitable for temperature sensitive molecules.
The presence of the trans-1,2-diaminocyclohexyl group in the newly synthesized ligand CyAAZTA led to the formation of a structurally rigid GaIII complex having good thermodynamic stability and kinetic inertness. These characteristics of Ga(CyAAZTA)- is well suited for 68Ga PET studies as decomposition takes considerably longer than the radioactive half-life of the 68Ga isotope. The complexation of 68Ga with CyAAZTA performed at RT and pH 3.8 within 15 min provide a yield of >80% and at 90°C with a near-quantitative yield (in 5 min), with high stability in HS and DTPA excess at 37°C. The potential of the proposed chelator is strenghtened by the wide spectrum of possible CyAAZTA-based bifunctional chelators that can be prepared by adapting procedures used for the syntheses of functionalized AAZTA ligands. Therefore, room temperature labelling can be of interest for temperature sensitive molecules and labelling at higher temperatures may be advantageous for reaching higher specific activity in peptide radiopharmaceuticals.
Chelators for Al18F labelling of biomolecules for PET-diagnosis
Pentadentate chelators able to stably complexed [Al18F]2+, thanks to their rigid structure, and to be marked with 18F very fastly at room temperature. The direct labelling at low temperature allows the diagnosis of several pathologies, thanks to the further conjugation of the chelators with sensible biomolecules, as proteins, anticorporal fragments, nanobodies, scFv.
Figure legend: Cartoon showing diverse possible application for temperature sensitive molecules radiolabeling using the new pentadentate chelator complexing [Al18F]2+.
The presence of heterocyclic or polycyclic structures and of five donator atoms, two neutral N-amminics and three negative O-carboxylic or phenolic, allows to obtain a fast and stable complexation also at room temperature. Moreover, the labelled target demonstrated a good stability in physiological conditions and in vivo, a low 18F accumulation into the bones and a fast hepatobiliary depletion. These prerogatives allow the development of bi-functional chelators able to conjugate a wide range of specific biomolecules for the pathologies determination. The Al18F labelling technique could allow the early diagnosis of oncological diseases and/or the identification of specific pathology at the initial stage. A wide number of applications of the bi-functional chelators with several selective biomolecules can be envisaged to identify pathologies detected with PET-imaging technique.
Semi rigid chelators for 89Zr labelling of biomolecules
Due to the fact that immunoPET has become the method of choice for imaging not only tumors but also immune cells, immune checkpoints, and inflammatory processes, the radiochemistry of Zr-89 and complexation strategies to use this radioisotope has driven the design and development of new chelators. We have recently developed and investigated hexadentate chelators for zirconium-89 starting from the heterocyclic structure of the triamine AMPED by insertion of three bidentate N-methylhydroxamate coordinating groups. The initial hypothesis was that longer pendant arms would better coordinate the Zr-89 isotope, leading to a more stable complex over time. A functionalized derivative of the most promising one was also synthesized and characterized to allow the conjugation with a humanized antibody and the subsequent 89Zr labeling.
Figure legend: Coordination scheme of 89Zr from the semi rigid AMPED-based chelator.
We selected the chelator with longer arms, AAZTHAG, as the best complexing agent for 89Zr presenting a stability of 86.4 ± 5.5% in human serum (HS) for at least 72 h. An activated ester functionalized version of AAZTHAG was synthesized to allow the conjugation with biomolecules such as human anti-HER2 monoclonal antibody Trastuzumab (Tz), as a proof of principle test. The final 89Zr labeled compound was characterized via radio-HPLC and SDS-PAGE followed by autoradiography, and its stability in different solutions confirmed for at least 4 days.