ABSTRACT
CD73 is a cell-surface ectoenzyme that hydrolyzes the conversion of extracellular adenosine monophosphate to adenosine, which in turn can promote resistance to immune checkpoint blockade therapy. Immune response may therefore be improved by targeting tumor CD73, and this possibility underlines the need to non-invasively assess tumor CD73 level. In this study, we developed a cysteine site-specific 89Zr-labeled anti-CD73 (89Zr-CD73) IgG immuno-PET technique that can image tumor CD73 expression in living bodies. Anti-CD73 IgG was reduced with tris(2-carboxyethyl)phosphine, underwent sulfohydryl moiety-specific conjugation with deferoxamine-maleimide, and was radiolabeled with 89Zr. CT26 mouse colon cancer cells, CT26/CD73 cells engineered to constitutively overexpress CD73, and 4T1.2 mouse breast cancer cells underwent cell binding assays and western blotting. Balb/c nude mice bearing tumors underwent 89Zr-CD73 IgG PET imaging and biodistribution studies. 89Zr-CD73 IgG showed 20-fold higher binding to overexpressing CT26/CD73 cells compared to low-expressing CT26 cells, and moderate expressing 4T1.2 cells showed uptake that was 38.9 ± 1.51% of CT26/CD73 cells. Uptake was dramatically suppressed by excess unlabeled antibody. CD73 content proportionately increased in CT26 and CT26/CD73 cell mixtures was associated with linear increases in 89Zr-CD73 IgG uptake. 89Zr-CD73 IgG PET/CT displayed clear accumulation in CT26/CD73 tumors with greater uptake compared to CT26 tumors (3.13 ± 1.70%ID/g vs. 1.27 ± 0.31%ID/g at 8 days; P = 0.04). Specificity was further supported by low CT26/CD73 tumor-to-blood ratio of 89Zr-isotype-IgG compared to 89Zr-CD73 IgG (0.48 ± 0.08 vs. 2.68 ± 0.52 at 4 days and 0.53 ± 0.07 vs. 4.81 ± 1.02 at 8 days; both P < 0.001). Immunoblotting and immunohistochemistry confirmed strong CD73 expression in CT26/CD73 tumors and low expression in CT26 tumors. 4T1.2 tumor mice also showed clear 89Zr-CD73 IgG accumulation at 8 days (3.75 ± 0.70%ID/g) with high tumor-to-blood ratio compared to 89Zr-isotype-IgG (4.91 ± 1.74 vs. 1.20 ± 0.28; P < 0.005). 89Zr-CD73 IgG specifically targeted CD73 on high expressing cancer cells in vitro and tumors in vivo. Thus, 89Zr-CD73 IgG immuno-PET may be useful for the non-invasive monitoring of CD73 expression in tumors of living subjects.
Subject(s)
5'-Nucleotidase , Colonic Neoplasms , Cysteine , Positron-Emission Tomography , Zirconium , Animals , 5'-Nucleotidase/metabolism , Zirconium/chemistry , Colonic Neoplasms/diagnostic imaging , Colonic Neoplasms/metabolism , Colonic Neoplasms/immunology , Colonic Neoplasms/pathology , Mice , Cell Line, Tumor , Positron-Emission Tomography/methods , Cysteine/metabolism , Humans , Radioisotopes , Female , Mice, Inbred BALB C , Tissue Distribution , Mice, Nude , GPI-Linked Proteins/metabolism , GPI-Linked Proteins/immunology , Immunoglobulin G/immunology , Immunoglobulin G/metabolismABSTRACT
Immunotherapy has transformed cancer treatment. Nevertheless, given the heterogeneity of clinical efficacy, the multiplicity of treatment options available and the possibility of serious adverse effects, selecting the most effective treatment has become the greatest challenge. Molecular imaging offers an attractive way for this purpose. ImmunoPET provides specific imaging with positron emission tomography (PET) using monoclonal antibodies (mAb) or its fragments as vector. By combining the high targeting specificity of mAb and the sensitivity of PET technique, immunoPET could noninvasively and dynamically reveal tumor antigens expression and provide theranostic tools of several types of malignancies. Because of their slow kinetics, mAbs require radioelements defined by a consistent half-life. Zirconium 89 (89Zr) and Copper 64 (64Cu) are radiometals with half-lives suitable for mAb labeling. Radiolabeling with a radiometal requires the prior use of a bifunctional chelate agent (BFCA) to functionalize mAb for radiometal chelation, in a second step. There are a number of BFCA available and much research is focused on antibody functionalization techniques or on developing the optimum chelating agent depending the selected radiometal. In this manuscript, we present a critical account of radiochemical techniques with radionuclides 89Zr and 64Cu and their applications in preclinical and clinical immuno-PET imaging.
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PURPOSE: Immune cells are capable of eliminating leukemic cells, as evidenced by outcomes in hematopoietic cell transplantation (HCT). However, patients who fail induction therapy will not benefit from HCT due to their minimal residual disease (MRD) status. Thus, we aimed to develop an immunomodulatory agent to reduce MRD by activating immune effector cells in the presence of leukaemia cells via a novel fusion protein that chimerises two clinically tolerated biologics: a CD33 antibody and the IL15Ra/IL15 complex (CD33xIL15). METHODS: We generated a set of CD33xIL15 fusion protein constructs with varying configurations and identified those with the best in vitro AML-binding, T cell activation, and NK cell potentiation. Using 89Zr-immunoPET imaging we then evaluated the biodistribution and in vivo tumour retention of the most favourable CD33xIL15 constructs in an AML xenograft model. Ex vivo biodistribution studies were used to confirm the pharmacokinetics of the constructs. RESULTS: Two of the generated fusion proteins, CD33xIL15 (N72D) and CD33xIL15wt, demonstrated optimal in vitro behaviour and were further evaluated in vivo. These studies revealed that the CD33xIL15wt candidate was capable of being retained in the tumour for as long as its parental CD33 antibody, Lintuzumab (13.9 ± 3.1%ID/g vs 18.6 ± 1.1%ID/g at 120 h). CONCLUSION: This work demonstrates that CD33xIL15 fusion proteins are capable of targeting leukemic cells and stimulating local T cells in vitro and of concentrating in the tumour in AML xenografts. It also highlights the importance of 89Zr-immunoPET to guide the development and selection of tumour-targeted antibody-cytokine fusion proteins.
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Purpose: Glypican-3 (GPC3)-targeted radioisotope immuno-positron emission tomography (immunoPET) may lead to earlier and more accurate diagnosis of hepatocellular carcinoma (HCC), thus facilitating curative treatment, decreasing early recurrence, and enhancing patient survival. We previously demonstrated reliable HCC detection using a zirconium-89-labeled murine anti-GPC3 antibody (89Zr-αGPC3M) for immunoPET. This study evaluated the efficacy of the humanized antibody successor (αGPC3H) to further clinical translation of a GPC3-based theranostic for HCC. Methods: In vitro αGPC3 binding to HepG2 cells was assessed by flow cytometry. In vivo 89Zr-αGPC3H and 89Zr-αGPC3M tumor uptake was evaluated by PET/CT and biodistribution studies in an orthotopic xenograft mouse model of HCC. Results: αGPC3H maintained binding to GPC3 in vitro and 89Zr-αGPC3H immunoPET identified liver tumors in vivo. PET/CT and biodistribution analyses demonstrated high 89Zr-αGPC3H tumor uptake and tumor-to-liver ratios, with no difference between groups. Conclusion: Humanized αGPC3 successfully targeted GPC3 in vitro and in vivo. 89Zr-αGPC3H immunoPET had comparable tumor detection to 89Zr-αGPC3M, with highly specific tumor uptake, making it a promising strategy to improve HCC detection.
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Lymphocyte activation gene 3 (LAG-3) has attracted much attention as a potentially valuable immune checkpoint. Individual identification of LAG-3 expression at screening and during treatment could improve the successful implementation of anti-LAG-3 therapies. HuL13 is a human IgG1 monoclonal antibody that binds to the LAG-3 receptor in T cells. Here, we used [89Zr]Zr-labeled HuL13 to delineate LAG-3+ T-cell infiltration into tumors via positron emission tomography (PET) imaging. A549/LAG-3 cells, which stably express LAG-3, were generated by infection with lentivirus. The uptake of [89Zr]Zr-DFO-HuL13 in A549/LAG-3 cells was greater than that in the negative control (A549/NC) cells at each time point. The equilibrium dissociation constant (Kd) of [89Zr]Zr-DFO-HuL13 for the LAG-3 receptor was 8.22 nM. PET imaging revealed significant uptake in the tumor areas of A549/LAG-3 tumor-bearing mice from 24 h after injection (SUVmax = 2.43 ± 0.06 at 24 h). As a proof of concept, PET imaging of the [89Zr]Zr-DFO-HuL13 tracer was further investigated in an MC38 tumor-bearing humanized LAG-3 mouse model. PET imaging revealed that the [89Zr]Zr-DFO-HuL13 tracer specifically targets human LAG-3 expressed on tumor-infiltrating lymphocytes (TILs). In addition to the tumors, the spleen was also noticeably visible. Tumor uptake of the [89Zr]Zr-DFO-HuL13 tracer was lower than its uptake in the spleen, but high uptake in the spleen could be reduced by coinjection of unlabeled antibodies. Coinjection of unlabeled antibodies increases tracer activity in the blood pool, thereby improving tumor uptake. Dosimetry evaluation of the healthy mouse models revealed that the highest absorbed radiation dose was in the spleen, followed by the liver and heart wall. In summary, these studies demonstrate the feasibility of using the [89Zr]Zr-DFO-HuL13 tracer for the detection of LAG-3 expression on TILs. Further clinical evaluation of the [89Zr]Zr-DFO-HuL13 tracer may be of significant help in the stratification and management of patients suitable for anti-LAG-3 therapy.
Subject(s)
Lymphocyte Activation Gene 3 Protein , Lymphocytes, Tumor-Infiltrating , Positron-Emission Tomography , Zirconium , Animals , Humans , Mice , Zirconium/chemistry , Lymphocytes, Tumor-Infiltrating/immunology , Lymphocytes, Tumor-Infiltrating/metabolism , Positron-Emission Tomography/methods , Cell Line, Tumor , Antigens, CD/metabolism , Antigens, CD/immunology , Radioisotopes/chemistry , Antibodies, Monoclonal/chemistry , Female , Tissue DistributionABSTRACT
Introduction: This study aimed to investigate the dynamics of programmed death-ligand 1 (PD-L1) expression, spatial heterogeneity, and binding affinity of FDA-approved anti-PD-L1 antibodies (avelumab and atezolizumab) in gastric cancer. Additionally, we determined how PD-L1 glycosylation impacts antibody accumulation in gastric cancer cells. Methods: Dynamic PD-L1 expression was examined in NCIN87 gastric cancer cells. Comparative binding studies of avelumab and atezolizumab were conducted in gastric cancer models, both in vitro and in vivo. Antibody uptake in tumors was visualized through positron emission tomography (PET) imaging. PD-L1 glycosylation status was determined via Western blot analyses before and after PNGase F treatment. Results: Consistent findings revealed time-dependent PD-L1 induction in NCIN87 gastric cancer cells and spatial heterogeneity in tumors, as shown by PET imaging and immunofluorescence. Avelumab displayed superior binding affinity to NCIN87 cells compared to atezolizumab, confirmed by in vivo PET imaging and ex vivo biodistribution analyses. Notably, PD-L1 glycosylation at approximately 50 kDa was observed, with PNGase F treatment inducing a shift to 35 kDa in molecular weight. Tissue samples from patient-derived xenografts (PDXs) validated the presence of both glycosylated and deglycosylated PD-L1 (degPD-L1) forms in gastric cancer. Immunofluorescence microscopy and binding assays demonstrated enhanced avelumab binding post-deglycosylation. Discussion: This study provides an understanding of dynamic and spatially heterogeneous PD-L1 expression in gastric cancer. Anti-PD-L1 immunoPET was able to visualize gastric tumors, and PD-L1 glycosylation has significant implications for antibody recognition. These insights contribute to demonstrating the complexities of PD-L1 in gastric cancer, holding relevance for refining PD-L1 imaging-based approaches.
Subject(s)
Antibodies, Monoclonal, Humanized , B7-H1 Antigen , Stomach Neoplasms , Stomach Neoplasms/metabolism , Stomach Neoplasms/immunology , Stomach Neoplasms/diagnostic imaging , B7-H1 Antigen/metabolism , Humans , Animals , Mice , Cell Line, Tumor , Glycosylation , Antibodies, Monoclonal, Humanized/therapeutic use , Xenograft Model Antitumor Assays , Female , Positron-Emission TomographyABSTRACT
BACKGROUND: Immune-positron emission tomography (PET) imaging with tracers that target CD8 and granzyme B has shown promise in predicting the therapeutic response following immune checkpoint blockade (ICB) in immunologically "hot" tumors. However, immune dynamics in the low T-cell infiltrating "cold" tumor immune microenvironment during ICB remain poorly understood. This study uses molecular imaging to evaluate changes in CD4 + T cells and CD8 + T cells during ICB in breast cancer models and examines biomarkers of response. METHODS: [89Zr]Zr-DFO-CD4 and [89Zr]Zr-DFO-CD8 radiotracers were used to quantify changes in intratumoral and splenic CD4 T cells and CD8 T cells in response to ICB treatment in 4T1 and MMTV-HER2 mouse models, which represent immunologically "cold" tumors. A correlation between PET quantification metrics and long-term anti-tumor response was observed. Further biological validation was obtained by autoradiography and immunofluorescence. RESULTS: Following ICB treatment, an increase in the CD8-specific PET signal was observed within 6 days, and an increase in the CD4-specific PET signal was observed within 2 days in tumors that eventually responded to immunotherapy, while no significant differences in CD4 or CD8 were found at the baseline of treatment that differentiated responders from nonresponders. Furthermore, mice whose tumors responded to ICB had a lower CD8 PET signal in the spleen and a higher CD4 PET signal in the spleen compared to non-responders. Intratumoral spatial heterogeneity of the CD8 and CD4-specific PET signals was lower in responders compared to non-responders. Finally, PET imaging, autoradiography, and immunofluorescence signals were correlated when comparing in vivo imaging to ex vivo validations. CONCLUSIONS: CD4- and CD8-specific immuno-PET imaging can be used to characterize the in vivo distribution of CD4 + and CD8 + T cells in response to immune checkpoint blockade. Imaging metrics that describe the overall levels and distribution of CD8 + T cells and CD4 + T cells can provide insight into immunological alterations, predict biomarkers of response to immunotherapy, and guide clinical decision-making in those tumors where the kinetics of the response differ.
Subject(s)
Breast Neoplasms , CD4-Positive T-Lymphocytes , CD8-Positive T-Lymphocytes , Disease Models, Animal , Immune Checkpoint Inhibitors , Positron-Emission Tomography , Tumor Microenvironment , Animals , Tumor Microenvironment/immunology , Female , Mice , CD8-Positive T-Lymphocytes/immunology , Immune Checkpoint Inhibitors/therapeutic use , Immune Checkpoint Inhibitors/pharmacology , CD4-Positive T-Lymphocytes/immunology , CD4-Positive T-Lymphocytes/metabolism , Positron-Emission Tomography/methods , Breast Neoplasms/diagnostic imaging , Breast Neoplasms/immunology , Breast Neoplasms/drug therapy , Breast Neoplasms/pathology , Breast Neoplasms/metabolism , Breast Neoplasms/therapy , Humans , Lymphocytes, Tumor-Infiltrating/immunology , Lymphocytes, Tumor-Infiltrating/metabolism , Cell Line, Tumor , Zirconium , Radiopharmaceuticals , RadioisotopesABSTRACT
BACKGROUND: Regadenoson, an agonist of adenosine A2 receptors, enables transient blood-brain barrier (BBB) disruption. The relevance of regadenoson as a pharmacological strategy for brain delivery was investigated using in vivo PET imaging in rats. RESEARCH DESIGN AND METHODS: Kinetic modeling of brain PET data was performed to estimate the impact of regadenoson (0.05 mg.kg-1, i.v.) on BBB permeation compared with control rats (n = 4-6 per group). Three radiolabeled compounds of different sizes, which do not cross the intact BBB, were tested. RESULTS: Regadenoson significantly increased the BBB penetration (+116 ± 13%, p < 0.001) of [18F]2-deoxy-2-fluoro-D-sorbitol ([18F]FDS, MW = 183 Da), a small-molecule marker of BBB permeability. The magnitude of the effect was different across brain regions, with a maximum increase in the striatum. Recovery of BBB integrity was observed 30 min after regadenoson injection. Regadenoson also increased the brain penetration (+72 ± 45%, p < 0.05) of a radiolabeled nanoparticle [89Zr]AGuIX (MW = 9 kDa). However, the brain kinetics of a monoclonal antibody ([89Zr]mAb, MW = 150 kDa) remained unchanged (p > 0.05). CONCLUSIONS: PET imaging showed the features and limitations of BBB disruption induced by regadenoson in terms of extent, regional distribution, and reversibility. Nevertheless, regadenoson enables the brain delivery of small molecules or nanoparticles in rats.
Subject(s)
Adenosine A2 Receptor Agonists , Blood-Brain Barrier , Brain , Positron-Emission Tomography , Purines , Pyrazoles , Animals , Blood-Brain Barrier/metabolism , Blood-Brain Barrier/drug effects , Purines/pharmacology , Purines/administration & dosage , Purines/pharmacokinetics , Pyrazoles/pharmacology , Pyrazoles/administration & dosage , Pyrazoles/pharmacokinetics , Rats , Positron-Emission Tomography/methods , Brain/metabolism , Brain/diagnostic imaging , Brain/drug effects , Male , Adenosine A2 Receptor Agonists/pharmacology , Adenosine A2 Receptor Agonists/administration & dosage , Drug Delivery Systems , Nanoparticles , Rats, Sprague-Dawley , Permeability , Fluorine Radioisotopes , Rats, WistarABSTRACT
Radiotheranostics is a rapidly growing approach in personalized medicine, merging diagnostic imaging and targeted radiotherapy to allow for the precise detection and treatment of diseases, notably cancer. Radiolabeled antibodies have become indispensable tools in the field of cancer theranostics due to their high specificity and affinity for cancer-associated antigens, which allows for accurate targeting with minimal impact on surrounding healthy tissues, enhancing therapeutic efficacy while reducing side effects, immune-modulating ability, and versatility and flexibility in engineering and conjugation. However, there are inherent limitations in using antibodies as a platform for radiopharmaceuticals due to their natural activities within the immune system, large size preventing effective tumor penetration, and relatively long half-life with concerns for prolonged radioactivity exposure. Antibody engineering can solve these challenges while preserving the many advantages of the immunoglobulin framework. In this review, the goal is to give a general overview of antibody engineering and design for tumor radiotheranostics. Particularly, the four ways that antibody engineering is applied to enhance radioimmunoconjugates: pharmacokinetics optimization, site-specific bioconjugation, modulation of Fc interactions, and bispecific construct creation are discussed. The radionuclide choices for designed antibody radionuclide conjugates and conjugation techniques and future directions for antibody radionuclide conjugate innovation and advancement are also discussed.
Subject(s)
Neoplasms , Radioimmunotherapy , Humans , Neoplasms/immunology , Neoplasms/radiotherapy , Neoplasms/therapy , Radioimmunotherapy/methods , Radiopharmaceuticals/therapeutic use , Animals , Immunoconjugates/chemistry , Protein Engineering/methodsABSTRACT
A key aspect for the applicability of 89Zr-radioimmunoconjugates is inert modification and radiolabeling. The two commercially available bifunctional variants of the siderophore desferrioxamine (DFO), Fe-DFO-N-suc-TFP-ester and p-NCS-Bz-DFO, are most often used for clinical 89Zr-immuno-PET. The use of Fe-DFO-N-suc-TFP-ester is advantageous with regard to higher radiolysis stability and more facile assessment of radiochemical purity as well as chelator-to-mAb ratio. However, not all mAbs withstand the Fe-removal step at relatively low pH (4-4.5) using EDTA, which is needed after conjugation to allow 89Zr labeling. In this study, it was investigated whether hydroxybenzyl ethylenediamine (HBED) or the clinically approved deferiprone (DFP) can serve as an alternative for EDTA to establish a pH-independent mild method for Fe-removal and thereby broaden the applicability of Fe-DFO-N-suc-TFP-ester. Carrier-added [59Fe]Fe-DFO-N-suc-TFP-ester was used for mAb modification to enable direct tracking of the Fe-removal efficiency under various conditions. Whereas incomplete Fe-removal with HBED was observed at pH 5 or higher, Fe-removal with DFP was possible at a broad pH range (4-9). This provides a mild, pH-independent method for Fe-removal, improving the applicability and attractiveness of Fe-DFO-N-suc-TFP-ester for 89Zr-mAb preparation.
Subject(s)
Deferoxamine , Iron , Positron-Emission Tomography , Radioisotopes , Zirconium , Zirconium/chemistry , Deferoxamine/chemistry , Radioisotopes/chemistry , Iron/chemistry , Positron-Emission Tomography/methods , Pyridones/chemistry , Deferiprone/chemistry , Immunoconjugates/chemistry , Radiopharmaceuticals/chemistry , Antibodies, Monoclonal/chemistryABSTRACT
BACKGROUND: During the previous two decades, PET imaging of biopharmaceuticals radiolabeled with zirconium-89 has become a consistent tool in preclinical and clinical drug development and patient selection, primarily due to its advantageous physical properties that allow straightforward radiolabeling of antibodies (89Zr-immuno-PET). The extended half-life of 78.4 h permits flexibility with respect to the logistics of tracer production, transportation, and imaging and allows imaging at later points in time. Additionally, its relatively low positron energy contributes to high-sensitivity, high-resolution PET imaging. Considering the growing interest in radiolabeling antibodies, antibody derivatives, and other compound classes with 89Zr in both clinical and pre-clinical settings, there is an urgent need to acquire valuable recommendations and guidelines towards standardization of labeling procedures. MAIN BODY: This review provides an overview of the key aspects of 89Zr-radiochemistry and radiopharmaceuticals. Production of 89Zr, conjugation with the mostly used chelators and radiolabeling strategies, and quality control of the radiolabeled products are described in detail, together with discussions about alternative options and critical steps, as well as recommendations for troubleshooting. Moreover, some historical background on 89Zr-immuno-PET, coordination chemistry of 89Zr, and future perspectives are provided. This review aims to serve as a quick-start guide for scientists new to the field of 89Zr-immuno-PET and to suggest approaches for harmonization and standardization of current procedures. CONCLUSION: The favorable PET imaging characteristics of 89Zr, its excellent availability due to relatively simple production and purification processes, and the development of suitable bifunctional chelators have led to the widespread use of 89Zr. The combination of antibodies and 89Zr, known as 89Zr-immuno-PET, has become a cornerstone in drug development and patient selection in recent years. Despite the advanced state of 89Zr-immuno-PET, new developments in chelator conjugation and radiolabeling procedures, application in novel compound classes, and improved PET scanner technology and quantification methods continue to reshape its landscape towards improving clinical outcomes.
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The incidence of androgen receptor (AR)-negative (AR-) prostate cancer, including aggressive neuroendocrine prostate cancer (NEPC), has more than doubled in the last decade, but its timely diagnosis is difficult as it lacks typical prostate cancer hallmarks. The carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5) has recently been identified as an upregulated surface antigen in NEPC. We developed an immuno-PET agent targeting CEACAM5 and evaluated its ability to delineate AR- prostate cancer in vivo. Methods: CEACAM5 expression was evaluated in a panel of prostate cancer cell lines by immunohistochemistry and Western blotting. The CEACAM5-targeting antibody labetuzumab was conjugated with the chelator desferrioxamine (DFO) and radiolabeled with 89Zr. The in vivo distribution of the radiolabeled antibody was evaluated in xenograft prostate cancer models by PET imaging and ex vivo organ distribution. Results: The NEPC cell line H660 exhibited strong CEACAM5 expression, whereas expression was limited in the AR- cell lines PC3 and DU145 and absent in the AR-positive cell line LNCaP. [89Zr]Zr-DFO-labetuzumab imaging was able to clearly delineate both neuroendocrine H660 xenografts and AR- DU145 in vivo but could not detect the AR-positive xenograft LNCaP. Conclusion: Immuno-PET imaging with [89Zr]Zr-DFO-labetuzumab is a promising diagnostic tool for AR- prostate cancer.
Subject(s)
GPI-Linked Proteins , Positron-Emission Tomography , Prostatic Neoplasms , Receptors, Androgen , Male , Humans , Prostatic Neoplasms/diagnostic imaging , Prostatic Neoplasms/metabolism , Prostatic Neoplasms/pathology , Cell Line, Tumor , Animals , Mice , Receptors, Androgen/metabolism , GPI-Linked Proteins/metabolism , Antigens, CD/metabolism , Zirconium , Tissue Distribution , Cell Adhesion Molecules/metabolism , Radioisotopes , Carcinoembryonic AntigenABSTRACT
BACKGROUND: Positron emission tomography (PET) is a highly sensitive method that provides fine resolution images, useful in the field of clinical diagnostics. In this context, Zirconium-89 (89Zr)-based imaging agents have represented a great challenge in molecular imaging with immuno-PET, which employs antibodies (mAbs) as biological vectors. Indeed, immuno-PET requires radionuclides that can be attached to the mAb to provide stable in vivo conjugates, and for this purpose, the radioactive element should have a decay half-life compatible with the time needed for the biodistribution of the immunoglobulin. In this regard, 89Zr is an ideal radioisotope for immuno-PET because its half-life perfectly matches the in vivo pharmacokinetics of mAbs. RESULTS: The main objective of this work was the design and synthesis of a series of bifunctional octadentate pseudopeptides able to generate stable 89Zr complexes. To achieve this, here we investigated hydroxamate, N-methylhydroxamate and catecholate chelating moieties in complexing radioactive zirconium. N-methylhydroxamate proved to be the most effective 89Zr-chelating group. Furthermore, the increased flexibility and hydrophilicity obtained by using polyoxyethylene groups spacing the hydroxamate units led to chelators capable of rapidly forming (15 min) stable and water-soluble complexes with 89Zr under mild reaction conditions (aqueous environment, room temperature, and physiological pH) that are mandatory for complexation reactions involving biomolecules. Additionally, we report challenge experiments with the competitor ligand EDTA and metal ions such as Fe3+, Zn2+ and Cu2+. In all examined conditions, the chelators demonstrated stability against transmetallation. Finally, a maleimide moiety was introduced to apply one of the most promising ligands in bioconjugation reactions through Thiol-Michael chemistry. CONCLUSION: Combining solid phase and solution synthesis techniques, we identified novel 89Zr-chelating molecules with a peptide scaffold. The adopted chemical design allowed modulation of molecular flexibility, hydrophilicity, as well as the decoration with different zirconium chelating groups. Best results in terms of 89Zr-chelating properties were achieved with the N-methyl hydroxamate moiety. The Zirconium complexes obtained with the most effective compounds were water-soluble, stable to transmetallation, and resistant to peptidases for at least 6 days. Further studies are needed to assess the potential of this novel class of molecules as Zirconium-chelating agents for in vivo applications.
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Accurately predicting and selecting patients who can benefit from targeted or immunotherapy is crucial for precision therapy. Trophoblast cell surface antigen 2 (Trop2) has been extensively investigated as a pan-cancer biomarker expressed in various tumours and plays a crucial role in tumorigenesis through multiple signalling pathways. Our laboratory successfully developed two 68Ga-labelled nanobody tracers that can rapidly and specifically target Trop2. Of the two tracers, [68Ga]Ga-NOTA-T4, demonstrated excellent pharmacokinetics in preclinical mouse models and a beagle dog. Moreover, [68Ga]Ga-NOTA-T4 immuno-positron emission tomography (immunoPET) allowed noninvasive visualisation of Trop2 heterogeneous and differential expression in preclinical solid tumour models and ten patients with solid tumours. [68Ga]Ga-NOTA-T4 immunoPET could facilitate clinical decision-making through patient stratification and response monitoring during Trop2-targeted therapies.
Subject(s)
Antigens, Neoplasm , Cell Adhesion Molecules , Neoplasms , Positron-Emission Tomography , Animals , Dogs , Female , Mice , Antigens, Neoplasm/metabolism , Antigens, Neoplasm/immunology , Cell Adhesion Molecules/metabolism , Neoplasms/diagnostic imaging , Neoplasms/immunology , Positron-Emission Tomography/methods , Single-Domain Antibodies/immunologyABSTRACT
BACKGROUND: The lack of an efficient way to screen patients who are responsive to immunotherapy challenges PD1/CTLA4-targeting cancer treatment. Immunotherapeutic efficacy cannot be clearly determined by peripheral blood analyses, tissue gene markers or CT/MR value. Here, we used a radionuclide and imaging techniques to investigate the novel dual targeted antibody cadonilimab (AK104) in PD1/CTLA4-positive cells in vivo. METHODS: First, humanized PD1/CTLA4 mice were purchased from Biocytogen Pharmaceuticals (Beijing) Co., Ltd. to express hPD1/CTLA4 in T-cells. Then, mouse colon cancer MC38-hPD-L1 cell xenografts were established in humanized mice. A bispecific antibody targeting PD1/CTLA4 (AK104) was labeled with radio-nuclide iodine isotopes. Immuno-PET/CT imaging was performed using a bispecific monoclonal antibody (mAb) probe 124I-AK104, developed in-house, to locate PD1+/CTLA4+ tumor-infiltrating T cells and monitor their distribution in mice to evaluate the therapeutic effect. RESULTS: The 124I-AK104 dual-antibody was successfully constructed with ideal radiochemical characteristics, in vitro stability and specificity. The results of immuno-PET showed that 124I-AK104 revealed strong hPD1/CTLA4-positive responses with high specificity in humanized mice. High uptake of 124I-AK104 was observed not only at the tumor site but also in the spleen. Compared with PD1- or CTLA4-targeting mAb imaging, 124I-AK104 imaging had excellent standard uptake values at the tumor site and higher tumor to nontumor (T/NT) ratios. CONCLUSIONS: The results demonstrated the potential of translating 124I-AK104 into a method for screening patients who benefit from immunotherapy and the efficacy, as well as the feasibility, of this method was verified by immuno-PET imaging of humanized mice.
Subject(s)
Antibodies, Bispecific , CTLA-4 Antigen , Positron Emission Tomography Computed Tomography , Programmed Cell Death 1 Receptor , Animals , Antibodies, Bispecific/pharmacology , Antibodies, Bispecific/immunology , Humans , Mice , CTLA-4 Antigen/immunology , Cell Line, Tumor , Positron Emission Tomography Computed Tomography/methods , Programmed Cell Death 1 Receptor/immunology , Colonic Neoplasms/diagnostic imaging , Colonic Neoplasms/immunology , Colonic Neoplasms/drug therapy , Colonic Neoplasms/pathology , Iodine Radioisotopes , Xenograft Model Antitumor Assays , Tissue Distribution , Lymphocytes, Tumor-Infiltrating/immunology , Lymphocytes, Tumor-Infiltrating/drug effects , FemaleABSTRACT
PURPOSE: The cluster of differentiation (CD70) is a potential biomarker of clear cell renal cell carcinoma (ccRCC). This study aims to develop CD70-targeted immuno-positron emission tomography/computed tomography (immunoPET/CT) imaging tracers and explore the diagnostic value in preclinical studies and the potential value in detecting metastases in ccRCC patients. METHODS: Four novel CD70-specific single-domain antibodies (sdAbs) were produced and labelled with 18F by the aluminium fluoride restrained complexing agent (AlF-RESCA) method to develop radiotracers. The visualisation properties of the tracers were evaluated in a subcutaneous ccRCC patient-derived xenograft (PDX) model. In a registered prospective clinical trial (NCT06148220), six patients with pathologically confirmed RCC were included and underwent immunoPET/CT examination exploiting one of the developed tracers (i.e., [18F]RCCB6). RESULTS: We engineered four sdAbs (His-tagged RCCB3 and RCCB6, His-tag-free RB3 and RB6) specifically targeting recombinant human CD70 without cross-reactivity to murine CD70. ImmunoPET/CT imaging with [18F]RCCB3 and [18F]RCCB6 demonstrated a high tumour-to-background ratio in a subcutaneous ccRCC PDX model, with the latter showing better diagnostic potential supported by higher tumour uptake and lower bone accumulation. In comparison, [18F]RB6, developed by sequence optimisation, has significantly lower kidney accumulation than that of [18F]RCCB6. In a pilot translational study, [18F]RCCB6 immunoPET/CT displayed ccRCC metastases in multiple patients and demonstrated improved imaging contrast and diagnostic value than 18F-FDG PET/CT in a patient with ccRCC. CONCLUSION: The work successfully developed a series of CD70-targeted immunoPET/CT imaging tracers. Of them, [18F]RCCB6 clearly and specifically identified inoculated ccRCCs in preclinical studies. Clinical translation of [18F]RCCB6 suggests potential for identifying recurrence and/or metastasis in ccRCC patients.
Subject(s)
Carcinoma, Renal Cell , Kidney Neoplasms , Positron Emission Tomography Computed Tomography , Humans , Carcinoma, Renal Cell/diagnostic imaging , Positron Emission Tomography Computed Tomography/methods , Kidney Neoplasms/diagnostic imaging , Female , Male , Fluorine Radioisotopes/chemistry , Animals , Mice , Middle Aged , Single-Domain Antibodies , Aged , Cell Line, Tumor , Tissue DistributionABSTRACT
Breast cancer (BrCa) ranks as the most prevalent malignant neoplasm affecting women worldwide. The expression of programmed death-ligand 1 (PD-L1) in BrCa has recently emerged as a biomarker for immunotherapy response, but traditional immunohistochemistry (IHC)-based methods are hindered by spatial and temporal heterogeneity. Noninvasive and quantitative PD-L1 imaging using appropriate radiotracers can serve to determine PD-L1 expression in tumors. This study aims to demonstrate the viability of PET imaging with 64Cu-labeled Durvalumab (abbreviated as Durva) to assess PD-L1 expression using a murine xenograft model of breast cancer. Durvalumab, a human IgG1 monoclonal antibody against PD-L1, was assessed for specificity in vitro in two cancer cell lines (MDA-MB-231 triple-negative breast cancer cell line and AsPC-1 pancreatic cancer cell line) with positive and negative PD-L1 expression by flow cytometry. Next, we performed the in vivo evaluation of 64Cu-NOTA-Durva in murine models of human breast cancer by PET imaging and ex vivo biodistribution. Additionally, mice bearing AsPC-1 tumors were employed as a negative control. Tumor uptake was quantified based on a 3D region-of-interest (ROI) analysis of the PET images and ex vivo biodistribution measurements, and the results were compared against conventional IHC testing. The radiotracer uptake was evident in MDA-MB-231 tumors and showed minimal nonspecific binding, corroborating IHC-derived results. The results of the biodistribution showed that the MDA-MB-231 tumor uptake of 64Cu-NOTA-Durva was much higher than 64Cu-NOTA-IgG (a nonspecific radiolabeled IgG). In Conclusion, 64Cu-labeled Durvalumab PET/CT imaging offers a promising, noninvasive approach to evaluate tumor PD-L1 expression.
ABSTRACT
BACKGROUND: Cancer stem cells play an important role in driving tumor growth and treatment resistance, which makes them a promising therapeutic target to prevent cancer recurrence. Emerging cancer stem cell-targeted therapies would benefit from companion diagnostic imaging probes to aid in patient selection and monitoring response to therapy. To this end, zirconium-89-radiolabeled immunoPET probes that target the cancer stem cell-antigen CD133 were developed using fully human antibody and antibody scFv-Fc scaffolds. RESULTS: ImmunoPET probes [89Zr]-DFO-RW03IgG (CA = 0.7 ± 0.1), [89Zr]-DFO-RW03IgG (CA = 3.0 ± 0.3), and [89Zr]-DFO-RW03scFv - Fc (CA = 2.9 ± 0.3) were radiolabeled with zirconium-89 (radiochemical yield 42 ± 5%, 97 ± 2%, 86 ± 12%, respectively) and each was isolated in > 97% radiochemical purity with specific activities of 120 ± 30, 270 ± 90, and 200 ± 60 MBq/mg, respectively. In vitro binding assays showed a low-nanomolar binding affinity of 0.6 to 1.1 nM (95% CI) for DFO-RW03IgG (CA = 0.7 ± 0.1), 0.3 to 1.9 nM (95% CI) for DFO-RW03IgG (CA = 3.0 ± 0.3), and 1.5 to 3.3 nM (95% CI) for DFO-RW03scFv - Fc (C/A = 0.3). Biodistribution studies found that [89Zr]-DFO-RW03scFv - Fc (CA = 2.9 ± 0.3) exhibited the highest tumor uptake (23 ± 4, 21 ± 2, and 23 ± 4%ID/g at 24, 48, and 72 h, respectively) and showed low uptake (< 6%ID/g) in all off-target organs at each timepoint (24, 48, and 72 h). Comparatively, [89Zr]-DFO-RW03IgG (CA = 0.7 ± 0.1) and [89Zr]-DFO-RW03IgG (CA = 3.0 ± 0.3) both reached maximum tumor uptake (16 ± 3%ID/g and 16 ± 2%ID/g, respectively) at 96 h p.i. and showed higher liver uptake (10.2 ± 3%ID/g and 15 ± 3%ID/g, respectively) at that timepoint. Region of interest analysis to assess PET images of mice administered [89Zr]-DFO-RW03scFv - Fc (CA = 2.9 ± 0.3) showed that this probe reached a maximum tumor uptake of 22 ± 1%ID/cc at 96 h, providing a tumor-to-liver ratio that exceeded 1:1 at 48 h p.i. Antibody-antigen mediated tumor uptake was demonstrated through biodistribution and PET imaging studies, where for each probe, co-injection of excess unlabeled RW03IgG resulted in > 60% reduced tumor uptake. CONCLUSIONS: Fully human CD133-targeted immunoPET probes [89Zr]-DFO-RW03IgG and [89Zr]-DFO-RW03scFv - Fc accumulate in CD133-expressing tumors to enable their delineation through PET imaging. Having identified [89Zr]-DFO-RW03scFv - Fc (CA = 2.9 ± 0.3) as the most attractive construct for CD133-expressing tumor delineation, the next step is to evaluate this probe using patient-derived tumor models to test its detection limit prior to clinical translation.
ABSTRACT
Background: Delta-like ligand 3 (DLL3) is aberrantly expressed on the cell surface in many neuroendocrine cancers including small cell lung cancer (SCLC) and neuroendocrine prostate cancer (NEPC). Several therapeutic agents targeting DLL3 are in active clinical development. Molecular imaging of DLL3 would enable non-invasive diagnostic assessment to inform the use of DLL3-targeting therapeutics or to assess disease treatment response. Methods: We conducted a first-in-human immuno-positron emission tomography (immunoPET) imaging study of [89Zr]Zr-DFO-SC16.56, composed of the anti-DLL3 antibody SC16.56 conjugated to desferrioxamine (DFO) and the positron-emitting radionuclide zirconium-89, in 18 patients with neuroendocrine cancers. An initial cohort of three patients received 1-2 mCi of [89Zr]Zr-DFO-SC16.56 at a total mass dose of 2·5 mg and underwent serial PET and computed tomography (CT) imaging over the course of one week. Radiotracer clearance, tumor uptake, and radiation dosimetry were estimated. An expansion cohort of 15 additional patients were imaged using the initial activity and mass dose. Retrospectively collected tumor biopsies were assessed for DLL3 by immunohistochemistry (IHC) (n = 16). Findings: Imaging of the initial 3 SCLC patients demonstrated strong tumor-specific uptake of [89Zr]Zr-DFO-SC16.56, with similar tumor: background ratios at days 3, 4, and 7 post-injection. Serum clearance was bi-phasic with an estimated terminal clearance half-time of 119 h. The sites of highest background tracer uptake were blood pool and liver. The normal tissue receiving the highest radiation dose was liver; 1·8 mGy/MBq, and the effective dose was 0.49 mSv/MBq. Tumoral uptake varied both between and within patients, and across anatomic sites, with a wide range in SUVmax (from 3·3 to 66·7). Tumor uptake by [89Zr]Zr-DFO-SC16.56 was associated with protein expression in all cases. Two non-avid DLL3 NEPC cases by PET scanning demonstrated the lowest DLL3 expression by tumor immunohistochemistry. Only one patient had a grade 1 allergic reaction, while no grade ≥2 adverse events noted. Interpretation: DLL3 PET imaging of patients with neuroendocrine cancers is safe and feasible. These results demonstrate the potential utility of [89Zr]Zr-DFO-SC16.56 for non-invasive in vivo detection of DLL3-expressing malignancies. Funding: Supported by NIH R01CA213448 (JTP), R35 CA263816 (CMR), U24 CA213274 (CMR), R35 CA232130 (JSL), and a Prostate Cancer Foundation TACTICAL Award (JSL), Scannell foundation. The Radiochemistry and Molecular Imaging Probes Core Facility is supported by NIH P30 CA08748.
ABSTRACT
Radioimmunoconjugates targeting human epidermal growth factor receptor 2 (HER2) have shown potential to noninvasively visualize HER2-positive tumors. However, the stochastic approach that has been traditionally used to radiolabel these antibodies yields poorly defined and heterogeneous products with suboptimal in vivo performance. Here, we describe a first-in-human PET study on patients with HER2-positive breast cancer evaluating the safety, biodistribution, and dosimetry of 89Zr-site-specific (ss)-pertuzumab PET, a site-specifically labeled radioimmunoconjugate designed to circumvent the limitations of random stochastic lysine labeling. Methods: Six patients with HER2-positive metastatic breast cancer were enrolled in a prospective clinical trial. Pertuzumab was site-specifically modified with desferrioxamine (DFO) via a novel chemoenzymatic strategy and subsequently labeled with 89Zr. Patients were administered 74 MBq of 89Zr-ss-pertuzumab in 20 mg of total antibody intravenously and underwent PET/CT at 1 d, 3-4 d, and 5-8 d after injection. PET imaging, whole-body probe counts, and blood draws were performed to assess the pharmacokinetics, biodistribution, and dosimetry. Results: 89Zr-ss-pertuzumab PET/CT was used to assess HER2 status and heterogeneity to guide biopsy and decide the next line of treatment at progression. The radioimmunoconjugate was able to detect known sites of malignancy, suggesting that these tumor lesions were HER2-positive. The optimal imaging time point was 5-8 d after administration, and no toxicities were observed. Dosimetry estimates from OLINDA showed that the organs receiving the highest doses (mean ± SD) were kidney (1.8 ± 0.5 mGy/MBq), liver (1.7 ± 0.3 mGy/MBq), and heart wall (1.2 ± 0.1 mGy/MBq). The average effective dose for 89Zr-ss-pertuzumab was 0.54 ± 0.03 mSv/MBq, which was comparable to both stochastically lysine-labeled 89Zr-DFO-pertuzumab and 89Zr-DFO-trastuzumab. One patient underwent PET/CT with both 89Zr-ss-pertuzumab and 89Zr-DFO-pertuzumab 1 mo apart, with 89Zr-ss-pertuzumab demonstrating improved lesion detection and higher tracer avidity. Conclusion: This study demonstrated the safety, dosimetry, and potential clinical applications of 89Zr-ss-pertuzumab PET/CT. 89Zr-ss-pertuzumab may detect more lesions than 89Zr-DFO-pertuzumab. Potential clinical applications include real-time evaluation of HER2 status to guide biopsy and assist in treatment decisions.