RESUMO
For the past two decades, the emerging role of the endothelin (ET) axis in cancer has been extensively investigated, and its involvement in several mechanisms described as "hallmarks of cancer" has clearly highlighted its potential as a therapeutic target. Despite the growing interest in finding effective anticancer drugs, no breakthrough treatment has successfully made its way to the market. Recently, our team reported the development of a new immuno-positron emission tomography probe targeting the ET A receptor (ETA, one of the ET receptors) that allows the successful detection of ETA+ glioblastoma, paving the way for the elaboration of novel antibody-based strategies. In this study, we describe the synthesis of two PET/NIRF (positron emission tomography/near-infrared fluorescence) dually functionalized imaging agents, directed against ETA or ETB, that could be used to detect ET+ tumors and select patients that will be eligible for fluorescence-guided surgery. Both imaging modalities were brought together using a highly versatile tetrazine platform bearing the IRDye800CW fluorophore and desferrioxamine for 89Zr chelation. This so-called monomolecular multimodal imaging probe was then "clicked", via an inverse-electron-demand Diels-Alder reaction, to antibodies conjugated site-specifically with a trans-cyclooctene group. This approach has led to homogeneous and well-defined constructs that retained their high affinity and high specificity for their respective target, as shown by flow cytometry and NIRF in vivo imaging experiments in nude mice bearing CHO-ETA and CHO-ETB tumors. Ultimately, these bimodal immunoconjugates could be used to improve the outcomes of patients with ET+ tumors.
Assuntos
Glioblastoma , Imunoconjugados , Animais , Camundongos , Humanos , Receptores de Endotelina , Camundongos Nus , Tomografia por Emissão de Pósitrons/métodos , Imagem Óptica/métodos , Linhagem Celular TumoralRESUMO
BACKGROUND: The resistance of glioblastoma stem cells (GSCs) to treatment is one of the causes of glioblastoma (GBM) recurrence. Endothelin A receptor (ETA) overexpression in GSCs constitutes an attractive biomarker for targeting this cell subpopulation, as illustrated by several clinical trials evaluating the therapeutic efficacy of endothelin receptor antagonists against GBM. In this context, we have designed an immunoPET radioligand combining the chimeric antibody targeting ETA, chimeric-Rendomab A63 (xiRA63), with 89Zr isotope and evaluated the abilities of xiRA63 and its Fab (ThioFab-xiRA63) to detect ETA+ tumors in a mouse model xenografted orthotopically with patient-derived Gli7 GSCs. RESULTS: Radioligands were intravenously injected and imaged over time by µPET-CT imaging. Tissue biodistribution and pharmacokinetic parameters were analyzed, highlighting the ability of [89Zr]Zr-xiRA63 to pass across the brain tumor barrier and achieve better tumor uptake than [89Zr]Zr-ThioFab-xiRA63. CONCLUSIONS: This study shows the high potential of [89Zr]Zr-xiRA63 in specifically targeting ETA+ tumors, thus raising the possibility of detecting and treating ETA+ GSCs, which could improve the management of GBM patients.
Assuntos
Glioblastoma , Animais , Camundongos , Humanos , Glioblastoma/diagnóstico por imagem , Receptor de Endotelina A , Tomografia por Emissão de Pósitrons/métodos , Distribuição Tecidual , Anticorpos , Células-Tronco , Linhagem Celular Tumoral , ZircônioRESUMO
Among all approaches in molecular imaging, the combination of near-infrared fluorescence imaging (NIRF) with radioisotopic imaging (PET or SPECT) allows one to benefit from the advantages of each of the imaging techniques, which are very complementary and of comparable sensitivity. To this end, the construction of monomolecular multimodal probes (MOMIP) has made it possible to combine the two imaging modalities within the same molecule, thus limiting the number of bioconjugation sites and yielding more homogeneous conjugates compared with those prepared through sequential conjugation. However, in order to optimize the bioconjugation strategy and, at the same time, the pharmacokinetic and biodistribution properties of the resulting imaging agent, a site-specific approach may be preferred. To further investigate this hypothesis, random and glycan-based site-specific bioconjugation approaches were compared thanks to a SPECT/NIRF bimodal probe based on an aza-BODIPY fluorophore. The overall experiments conducted in vitro and in vivo on HER2-expressing tumors demonstrated a clear superiority of the site-specific approach to improve affinity, specificity, and biodistribution of the bioconjugates.
RESUMO
The human leucine-rich repeat-containing protein 15 (LRRC15) is a membrane protein identified as a marker of CAF (cancer-associated fibroblast) cells whose overexpression is positively correlated with cancer grade and outcome. Nuclear molecular imaging (i.e., SPECT and PET) to track LRRC15 expression could be very useful in guiding further therapeutic strategies. In this study, we developed an ScFv mouse phage-display library to obtain small fragment antibodies against human LRRC15 for molecular imaging purposes. Mice were immunized with recombinant human LRRC15 (hLRRC15), and lymph node cells were harvested for ScFv (single-chain variable fragment) phage-display analysis. The built library was used for panning on cell lines with constitutive or induced expression after transfection. The choice of best candidates was performed by screening various other cell lines, using flow cytometry. The selected candidates were reformatted into Cys-ScFv or Cys-diabody by addition of cysteine, and cloned in mammalian expression vectors to obtain batches of small fragments that were further used in site-specific radiolabeling tests. The obtained library was 1.2 × 107 cfu/µg with an insertion rate >95%. The two panning rounds performed on cells permittedenrichment of 2 × 10−3. Screening with flow cytometry allowed us to identify 28 specific hLRRC15 candidates. Among these, two also recognized murine LRCC15 and were reformatted into Cys-ScFv and Cys-diabody. They were expressed transiently in a mammalian system to obtain 1.0 to 4.5 mg of Cys fragments ready for bioconjugation and radiolabeling. Thus, in this paper, we demonstrate the relevance of the phage-display ScFv library approach for the fast-track development of small antibodies for imaging and/or immunotherapy purposes.
Assuntos
Bacteriófagos , Anticorpos de Cadeia Única , Humanos , Camundongos , Animais , Biblioteca de Peptídeos , Cisteína , Leucina , Ensaio de Imunoadsorção Enzimática , Proteínas de Membrana , Bacteriófagos/metabolismo , Mamíferos/metabolismoRESUMO
High expression levels of the tumor-associated antigen MUC1 have been correlated with tumor aggressiveness, poor response to therapy, and poor survival in several tumor types, including breast, pancreatic, and epithelial ovarian cancer. Herein, we report the synthesis, characterization, and in vivo evaluation of a novel radioimmunoconjugate for the immuno-positron emission tomography (immunoPET) imaging of MUC1 expression based on the AR20.5 antibody. To this end, we modified AR20.5 with the chelator desferrioxamine (DFO) and labeled it with the positron-emitting radiometal zirconium-89 (t1/2 ~3.3 d) to produce [89Zr]Zr-DFO-AR20.5. In subsequent in vivo experiments in athymic nude mice bearing subcutaneous MUC1-expressing ovarian cancer xenografts, [89Zr]Zr-DFO-AR20.5 clearly delineated tumor tissue, producing a tumoral activity concentration of 19.1 ± 6.4 percent injected dose per gram (%ID/g) at 120 h post-injection and a tumor-to-muscle activity concentration ratio of 42.4 ± 10.6 at the same time point. Additional PET imaging experiments in mice bearing orthotopic MUC1-expressing ovarian cancer xenografts likewise demonstrated that [89Zr]Zr-DFO-AR20.5 enables the visualization of tumor tissue-including metastatic lesions-with promising tumor-to-background contrast.
Assuntos
Anticorpos Monoclonais/química , Anticorpos Antineoplásicos/química , Imunoconjugados/química , Metástase Linfática/diagnóstico por imagem , Mucina-1/metabolismo , Neoplasias/diagnóstico por imagem , Radioisótopos/química , Zircônio/química , Animais , Anticorpos Monoclonais/metabolismo , Anticorpos Monoclonais/farmacocinética , Anticorpos Antineoplásicos/metabolismo , Disponibilidade Biológica , Desferroxamina/química , Feminino , Expressão Gênica , Xenoenxertos , Humanos , Imunoconjugados/metabolismo , Imunoconjugados/farmacocinética , Metástase Linfática/patologia , Camundongos , Camundongos Nus , Mucina-1/genética , Neoplasias/metabolismo , Neoplasias/patologia , Tomografia por Emissão de Pósitrons/métodos , Ligação Proteica , Razão Sinal-Ruído , Distribuição TecidualRESUMO
Rationale: The overwhelming majority of radioimmunoconjugates are produced via random conjugation methods predicated on attaching bifunctional chelators to the lysines of antibodies. However, this approach inevitably produces poorly defined and heterogeneous immunoconjugates because antibodies have several lysines distributed throughout their structure. To circumvent this issue, we have previously developed a chemoenzymatic bioconjugation strategy that site-specifically appends cargoes to the biantennary heavy chain glycans attached to CH2 domains of the immunoglobulin's Fc region. In the study at hand, we explore the effects of this approach to site-specific bioconjugation on the Fc receptor binding and in vivo behavior of radioimmunoconjugates. Methods: We synthesized three desferrioxamine (DFO)-labeled immunoconjugates based on the HER2-targeting antibody pertuzumab: one using random bioconjugation methods (DFO-nsspertuzumab) and two using variants of our chemoenzymatic protocol (DFO-sspertuzumab-EndoS and DFO-sspertuzumab-ßGal). Subsequently, we characterized these constructs and evaluated their ability to bind HER2, human FcγRI (huFcγRI), and mouse FcγRI (muFcγRI). After radiolabeling the immunoconjugates with zirconium-89, we conducted PET imaging and biodistribution studies in two different mouse models of HER2-expressing breast cancer. Results: MALDI-ToF and SDS-PAGE analysis confirmed the site-specific nature of the bioconjugation, and flow cytometry and surface plasmon resonance (SPR) revealed that all three immunoconjugates bind HER2 as effectively as native pertuzumab. Critically, however, SPR experiments also illuminated that DFO-sspertuzumab-EndoS possesses an attenuated binding affinity for huFcγRI (17.4 ± 0.3 nM) compared to native pertuzumab (4.7 ± 0.2 nM), DFO-nsspertuzumab (4.1 ± 0.1 nM), and DFO-sspertuzumab-ßGal (4.7 ± 0.2 nM). ImmunoPET and biodistribution experiments in athymic nude mice bearing HER2-expressing BT474 human breast cancer xenografts yielded no significant differences in the in vivo behavior of the radioimmunoconjugates. Yet experiments in tumor-bearing humanized NSG mice revealed that 89Zr-DFO-sspertuzumab-EndoS produces higher activity concentrations in the tumor (111.8 ± 39.9 %ID/g) and lower activity concentrations in the liver and spleen (4.7 ± 0.8 %ID/g and 13.1 ± 4.0 %ID/g, respectively) than its non-site-specifically labeled cousin, a phenomenon we believe stems from the altered binding of the former to huFcγRI. Conclusion: These data underscore that this approach to site-specific bioconjugation not only produces more homogeneous and well-defined radioimmunoconjugates than traditional methods but may also improve their in vivo performance in mouse models by reducing binding to FcγRI.
Assuntos
Neoplasias da Mama/metabolismo , Polissacarídeos/química , Compostos Radiofarmacêuticos/farmacocinética , Receptores de IgG/metabolismo , Animais , Anticorpos/efeitos dos fármacos , Anticorpos/imunologia , Anticorpos Monoclonais Humanizados , Antineoplásicos Imunológicos , Neoplasias da Mama/imunologia , Neoplasias da Mama/patologia , Desferroxamina/química , Feminino , Humanos , Imunoconjugados/química , Imunoconjugados/metabolismo , Imunoconjugados/farmacocinética , Camundongos , Camundongos Nus , Tomografia por Emissão de Pósitrons/métodos , Radioisótopos , Receptor ErbB-2/metabolismo , Receptores de IgG/química , Distribuição Tecidual , Ensaios Antitumorais Modelo de Xenoenxerto , ZircônioRESUMO
Antibodies are promising vectors for PET imaging. However, the high uptake of radioimmunoconjugates in nontarget tissues such as the liver and spleen hampers their performance as radiotracers. This off-target uptake can lead to suboptimal tumor-to-background activity concentration ratios, decreasing the contrast of images and reducing their diagnostic utility. A possible cause of this uptake is the sequestration of radioimmunoconjugates by immune cells bearing Fc-γ-receptors (FcγR) that bind to the Fc regions of antibodies. Methods: Since the heavy chain glycans influence the affinity of FcγR for the Fc domain, we set out to investigate whether radioimmunoconjugates with truncated glycans would exhibit altered binding to FcγRI and, in turn, improved in vivo performance. Using the HER2-targeting antibody trastuzumab, we synthesized a series of desferrioxamine-bearing immunoconjugates with differing glycosylation states and interrogated their FcγRI binding via surface plasmon resonance, enzyme-linked immunosorbent assay, and flow cytometry. Furthermore, we labeled these immunoconjugates with 89Zr and explored their biodistribution in athymic nude, NSG, and humanized NSG mice bearing human epidermal growth factor receptor 2-expressing human breast cancer xenografts. Results: We observed a strong correlation between the impaired in vitro FcγRI binding of deglycosylated immunoconjugates and significant decreases in the in vivo off-target uptake of the corresponding 89Zr-labeled radioimmunoconjugates (i.e., liver activity concentrations are reduced by â¼3.5-fold in humanized NSG mice). These reductions in off-target uptake were accompanied by concomitant increases in the tumoral activity concentrations of the glycoengineered radioimmunoconjugates, ultimately yielding improved tumor-to-healthy organ contrast and higher quality PET images. Conclusion: Our findings suggest that the deglycosylation of antibodies represents a facile strategy for improving the quality of immuno-PET in animal models as well as in certain patient populations.
Assuntos
Imunoconjugados/química , Tomografia por Emissão de Pósitrons , Receptores de IgG/química , Animais , Neoplasias da Mama/diagnóstico por imagem , Linhagem Celular Tumoral , Desferroxamina/química , Feminino , Glicosilação , Humanos , Imunoglobulina G/química , Técnicas In Vitro , Cinética , Camundongos , Camundongos Nus , Transplante de Neoplasias , Compostos Radiofarmacêuticos , Receptor ErbB-2/metabolismo , Proteínas Recombinantes/química , Ressonância de Plasmônio de Superfície , Distribuição Tecidual , Trastuzumab/química , Zircônio/químicaRESUMO
Over the past 25 years, antibodies have emerged as extraordinarily promising vectors for the delivery of radionuclides to tumors for nuclear imaging. While radioimmunoconjugates often produce very high activity concentrations in target tissues, they also are frequently characterized by elevated activity concentrations in healthy organs as well. The root of this background uptake lies in the complex network of biological interactions between the radioimmunoconjugate and the subject. In this review, we seek to provide an overview of these interactions and thus paint a general picture of the in vivo fate of radioimmunoconjugates. To cover the entire story, we have divided our discussion into 2 parts. First, we will address the path of the entire radioimmunoconjugate as it travels through the body. And second, we will cover the fate of the radionuclide itself, as its course can diverge from the antibody under certain circumstances. Ultimately, our goal is to provide the nuclear imaging field with a resource covering these important-yet often underestimated-pathways.
Assuntos
Imunoconjugados , Tomografia por Emissão de Pósitrons/métodos , Tomografia Computadorizada de Emissão de Fóton Único/métodos , Animais , Anticorpos/metabolismo , Humanos , Imunoconjugados/metabolismo , Imunoconjugados/farmacocinética , Radioisótopos , Distribuição TecidualRESUMO
The conjugation of antibodies with cytotoxic drugs can alter their in vivo pharmacokinetics. As a result, the careful assessment of the in vivo behavior, and specifically the tumor-targeting properties, of antibody-drug conjugates represents a crucial step in their development. In order to facilitate this process, we have created a methodology that facilitates the dual labeling of an antibody with both a toxin and a radionuclide for positron emission tomography (PET). To minimize the impact of these modifications, this chemoenzymatic approach leverages strain-promoted azide-alkyne click chemistry to graft both cargoes to the heavy chain glycans of the immuoglobulin's Fc domain. As a proof-of-concept, a HER2-targeting trastuzumab immunoconjugate was created bearing both a monomethyl auristatin E (MMAE) toxin as well as the long-lived positron-emitting radiometal 89Zr ( t1/2 ≈ 3.3 days). Both the tumor targeting and therapeutic efficacy of the 89Zr-trastuzumab-MMAE immunoconjugate were validated in vivo using a murine model of HER2-expressing breast cancer. The site-specifically dual-labeled construct enabled the clear visualization of tumor tissue via PET imaging, producing tumoral uptake of â¼70%ID/g. Furthermore, a longitudinal therapy study revealed that the immunoconjugate exerts significant antitumor activity, leading to a >90% reduction in tumor volume over the course of 20 days.
Assuntos
Anticorpos Monoclonais Humanizados/administração & dosagem , Antineoplásicos Imunológicos/administração & dosagem , Neoplasias da Mama/tratamento farmacológico , Imunoconjugados/administração & dosagem , Tomografia por Emissão de Pósitrons combinada à Tomografia Computadorizada/métodos , Animais , Anticorpos Monoclonais Humanizados/farmacocinética , Antineoplásicos Imunológicos/farmacocinética , Neoplasias da Mama/diagnóstico por imagem , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Química Click , Desenvolvimento de Medicamentos , Feminino , Humanos , Imunoconjugados/química , Imunoconjugados/farmacocinética , Camundongos , Camundongos Nus , Compostos Radiofarmacêuticos/administração & dosagem , Compostos Radiofarmacêuticos/química , Compostos Radiofarmacêuticos/farmacocinética , Receptor ErbB-2/antagonistas & inibidores , Distribuição Tecidual , Resultado do Tratamento , Carga Tumoral/efeitos dos fármacos , Microtomografia por Raio-X/métodosRESUMO
A critical benchmark in the development of antibody-based therapeutics is demonstration of efficacy in preclinical mouse models of human disease, many of which rely on immunodeficient mice. However, relatively little is known about how the biology of various immunodeficient strains impacts the in vivo fate of these drugs. Here we used immunoPET radiotracers prepared from humanized, chimeric, and murine mAbs against four therapeutic oncologic targets to interrogate their biodistribution in four different strains of immunodeficient mice bearing lung, prostate, and ovarian cancer xenografts. The immunodeficiency status of the mouse host as well as both the biological origin and glycosylation of the antibody contributed significantly to the anomalous biodistribution of therapeutic monoclonal antibodies in an Fc receptor-dependent manner. These findings may have important implications for the preclinical evaluation of Fc-containing therapeutics and highlight a clear need for biodistribution studies in the early stages of antibody drug development.Significance: Fc/FcγR-mediated immunobiology of the experimental host is a key determinant to preclinical in vivo tumor targeting and efficacy of therapeutic antibodies. Cancer Res; 78(7); 1820-32. ©2018 AACR.
Assuntos
Antineoplásicos Imunológicos/uso terapêutico , Cetuximab/uso terapêutico , Neoplasias Pulmonares/terapia , Neoplasias Ovarianas/terapia , Neoplasias da Próstata/terapia , Receptores Fc/imunologia , Receptores de IgG/imunologia , Trastuzumab/uso terapêutico , Animais , Linhagem Celular Tumoral , Modelos Animais de Doenças , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos Nus , Camundongos SCID , Transplante de Neoplasias , Tomografia por Emissão de Pósitrons/métodos , Imunodeficiência Combinada Severa/imunologia , Transplante HeterólogoRESUMO
BACKGROUND: Pretargeting-based approaches are being investigated for radioimmunoimaging and therapy applications to reduce the effective radiation burden to the patient. To date, only a few studies have used short-lived radioisotopes for pretargeting of antibodies, and such examples with internalizing antibodies are even rarer. Herein, we have investigated pretargeting methodology using inverse electron-demand Diels-Alder (IEDDA) for tracing two clinically relevant, internalizing monoclonal antibodies, cetuximab and trastuzumab. RESULTS: Bioorthogonal reaction between tetrazine and trans-cyclooctene (TCO) was used for tracing cetuximab and trastuzumab in vivo with a fluorine-18 (t ½ = 109.8 min) labelled tracer. TCO-cetuximab or TCO-trastuzumab was administered 24, 48, or 72 h prior to the injection of tracer to A431 or BT-474 tumour-bearing mice, respectively. With cetuximab, the highest tumour-to-blood ratios were achieved when the lag time between antibody and tracer injections was 72 h. With trastuzumab, no difference was observed between different lag times. For both antibodies, the tumour could be clearly visualized in the PET images with the highest tumour uptake of 3.7 ± 0.1%ID/g for cetuximab and 1.5 ± 0.1%ID/g for trastuzumab as quantified by ex vivo biodistribution. In vivo IEDDA reaction was observed in the blood for both antibodies, but with trastuzumab, this was to a much lower degree than with cetuximab. CONCLUSIONS: We could successfully visualize the tumours by using cetuximab and trastuzumab in pretargeted PET imaging despite the challenging circumstances where the antibody is internalized and there is still some unbound antibody circulating in the blood flow. This clearly demonstrates the potential of a pretargeted approach for targeting internalizing antigens and warrants development of pharmacokinetic optimization of the biorthogonal reactants to this end.