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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.
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Neoplasias da Mama , Linfócitos T CD4-Positivos , Linfócitos T CD8-Positivos , Modelos Animais de Doenças , Inibidores de Checkpoint Imunológico , Tomografia por Emissão de Pósitrons , Microambiente Tumoral , Animais , Microambiente Tumoral/imunologia , Feminino , Camundongos , Linfócitos T CD8-Positivos/imunologia , Inibidores de Checkpoint Imunológico/uso terapêutico , Inibidores de Checkpoint Imunológico/farmacologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Tomografia por Emissão de Pósitrons/métodos , Neoplasias da Mama/diagnóstico por imagem , Neoplasias da Mama/imunologia , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/patologia , Neoplasias da Mama/metabolismo , Neoplasias da Mama/terapia , Humanos , Linfócitos do Interstício Tumoral/imunologia , Linfócitos do Interstício Tumoral/metabolismo , Linhagem Celular Tumoral , Zircônio , Compostos Radiofarmacêuticos , RadioisótoposRESUMO
PURPOSE: The high expression of the transmembrane glycoprotein trophoblast cell-surface antigen 2 (Trop2) was strongly associated with the progression of solid tumors, including pancreatic and gastric cancers. Our study aimed to construct Trop2-specific immuno-positron emission tomography (immunoPET) probes and assess the diagnostic abilities in preclinical pancreatic and gastric cancer models. METHODS: The expression of Trop2 in pancreatic cancer was determined by single-cell sequencing and immunohistochemistry on tissue microarray (TMA). Flow cytometry was used to screen the expression of Trop2 in pancreatic cancer cell lines. Two nanobodies (i.e., RTD98 and RTD01) targeting Trop2 were developed and labeled with gallium-68 (68Ga, T1/2 = 1.1 h) to construct immunoPET imaging probes. The agents were researched in cell-derived pancreatic and patient-derived gastric cancer models expressing varying Trop2. RESULTS: Single-cell sequencing results showed high expression of Trop2 in pancreatic ductal cells as well as acinar cells and immunohistochemical staining of TMA from pancreatic cancers showed significantly higher expression of Trop2 in cancerous than in paracancerous tissues. ImmunoPET utilizing [68Ga]Ga-NOTA-RTD98 could clearly delineate subcutaneous tumors, both in cell-derived pancreatic cancer models and patient-derived gastric cancer models, superior to imaging using [18F]-FDG or a non-specific probe [68Ga]Ga-NOTA-RTD161. Another probe with improved pharmacokinetics targeting Trop2, [68Ga]Ga-NOTA-RTD01, was further prepared and showed advantageous diagnostic capabilities in preclinical pancreatic cancer models. CONCLUSION: In the work, we reported two nanobody tracers targeting human Trop2 which may facilitate better use of Trop2-targeted therapeutics by noninvasively displaying expression dynamics of the target.
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Neoplasias Pancreáticas , Neoplasias Gástricas , Humanos , Linhagem Celular Tumoral , Radioisótopos de Gálio , Imuno-Histoquímica , Neoplasias Pancreáticas/metabolismo , Tomografia por Emissão de Pósitrons/métodosRESUMO
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.
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Carcinoma de Células Renais , Neoplasias Renais , Tomografia por Emissão de Pósitrons combinada à Tomografia Computadorizada , Humanos , Carcinoma de Células Renais/diagnóstico por imagem , Tomografia por Emissão de Pósitrons combinada à Tomografia Computadorizada/métodos , Neoplasias Renais/diagnóstico por imagem , Feminino , Masculino , Radioisótopos de Flúor/química , Animais , Camundongos , Pessoa de Meia-Idade , Anticorpos de Domínio Único , Idoso , Linhagem Celular Tumoral , Distribuição TecidualRESUMO
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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Immune checkpoint inhibitors (ICIs) therapy based on programmed cell death ligand 1 (PD-L1) has shown significant development in treating several carcinomas, but not all patients respond to this therapy due to the heterogeneity of PD-L1 expression. The sensitive and accurate quantitative analysis of in vivo PD-L1 expression is critical for treatment decisions and monitoring therapy. In the present study, an aptamer-based dual-modality positron emission tomography/near-infrared fluorescence (PET/NIRF) imaging probe was developed, and its specificity and sensitivity to PD-L1 were assessed in vitro and in vivo. The probe precursor NOTA-Cy5-R1 was prepared by using automated solid-phase oligonucleotide synthesis. PET/NIRF dual-modality probe [68Ga]Ga-NOTA-Cy5-R1 was successfully synthesized and radiolabeled. The binding specificity of [68Ga]Ga-NOTA-Cy5-R1 to PD-L1 was evaluated by flow cytometry, fluorescence imaging, and cellular uptake in A375-hPD-L1 and A375 cells, and it showed good fluorescence properties and stability in vitro. In vivo PET/NIRF imaging studies illustrated that [68Ga]Ga-NOTA-Cy5-R1 can sensitively and specifically bind to PD-L1 positive tumors. Meanwhile, the rapid clearance of probes from nontarget tissues achieved a high signal-to-noise ratio. In addition, changes of PD-L1 expression in NCI-H1299 xenografts treated with cisplatin (CDDP) were sensitivity monitored by [68Ga]Ga-NOTA-Cy5-R1 PET imaging, and ex vivo autoradiography and western blot analyses correlated well with the change of PD-L1 expression in vivo. Overall, [68Ga]Ga-NOTA-Cy5-R1 showed notable potency as a dual-modality PET/NIRF imaging probe for visualizing tumors and monitoring the dynamic changes of PD-L1 expression, which can help to direct and promote the clinical practice of ICIs therapy.
Assuntos
Antígeno B7-H1 , Neoplasias , Humanos , Antígeno B7-H1/metabolismo , Radioisótopos de Gálio/química , Tomografia por Emissão de Pósitrons/métodos , Anticorpos , Neoplasias/diagnóstico por imagem , Neoplasias/tratamento farmacológico , Linhagem Celular TumoralRESUMO
Very late antigen-4 (VLA4; CD49d) is a promising immune therapy target in treatment-resistant leukemia and multiple myeloma, and there is growing interest in repurposing the humanized monoclonal antibody (Ab), natalizumab, for this purpose. Positron emission tomography with radiolabeled Abs (immuno-PET) could facilitate this effort by providing information on natalizumab's in vivo pharmacokinetic and target delivery properties. In this study, we labeled natalizumab with 89Zr specifically on sulfhydryl moieties via maleimide-deferoxamine conjugation. High VLA4-expressing MOLT4 human T cell acute lymphoblastic leukemia cells showed specific 89Zr-natalizumab binding that was markedly blocked by excess Ab. In nude mice bearing MOLT4 tumors, 89Zr-natalizumab PET showed high-contrast tumor uptake at 7 days postinjection. Biodistribution studies confirmed that uptake was the highest in MOLT4 tumors (2.22 ± 0.41%ID/g) and the liver (2.33 ± 0.76%ID/g), followed by the spleen (1.51 ± 0.42%ID/g), while blood activity was lower at 1.12 ± 0.21%ID/g. VLA4-specific targeting in vivo was confirmed by a 58.1% suppression of tumor uptake (0.93 ± 0.15%ID/g) when excess Ab was injected 1 h earlier. In cultured MOLT4 cells, short-term 3 day exposure to the proteasome inhibitor bortezomib (BTZ) did not affect the α4 integrin level, but BTZ-resistant cells that survived the treatment showed increased α4 integrin expression. When the effects of BTZ treatment were tested in mice, there was no change of the α4 integrin level or 89Zr-natalizumab uptake in MOLT4 leukemia tumors, which underscores the complexity of tumor VLA4 regulation in vivo. In conclusion, 89Zr-natalizumab PET may be useful for noninvasive monitoring of tumor VLA4 and may assist in a more rational application of Ab-based therapies for hematologic malignancies.
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Integrina alfa4beta1 , Leucemia , Humanos , Animais , Camundongos , Natalizumab/uso terapêutico , Cisteína , Integrina alfa4 , Camundongos Nus , Distribuição Tecidual , Linhagem Celular Tumoral , Tomografia por Emissão de Pósitrons/métodos , Zircônio/químicaRESUMO
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.
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Proteína do Gene 3 de Ativação de Linfócitos , Linfócitos do Interstício Tumoral , Tomografia por Emissão de Pósitrons , Zircônio , Animais , Humanos , Camundongos , Zircônio/química , Linfócitos do Interstício Tumoral/imunologia , Linfócitos do Interstício Tumoral/metabolismo , Tomografia por Emissão de Pósitrons/métodos , Linhagem Celular Tumoral , Antígenos CD/metabolismo , Antígenos CD/imunologia , Radioisótopos/química , Anticorpos Monoclonais/química , Feminino , Distribuição TecidualRESUMO
Extracellular matrix metalloproteinase inducer CD147 is a glycoprotein on the cell surface. There is minimal expression of CD147 in normal epithelial and fetal tissues, but it is highly expressed in a number of aggressive tumors. CD147 has been implicated in pan-cancer immunity and progression. With the development of CD147-targeting therapeutic strategy, accurate detection of CD147 expression in tumors and its changes during the therapy is necessary. In this study we constructed a novel radiotracer by labeling the anti-CD147 mAb with radionuclide 124/125I (124/125I-anti-CD147) for noninvasive detection of CD147 expression in pan-cancers, and characterized its physicochemical properties, affinity, metabolic characteristics, biodistribution and immunoPET imaging with 124I-IgG and 18F-FDG as controls. By examining the expression of CD147 in cancer cell lines, we found high CD147 expression in colon cancer cells LS174T, FADU human pharyngeal squamous cancer cells and 22RV1 human prostate cancer cells, and low expression of CD147 in human pancreatic cancer cells ASPC1 and human gastric cancer cells BGC823. 124/125I-anti-CD147 was prepared using N-bromine succinimide (NBS) as oxidant and purified by PD-10 column. Its radiochemical purity (RCP) was over 99% and maintained over 85% in saline or 5% human serum albumin (HSA) for more than 7 d; the RCP of 125I-anti-CD147 in blood was over 90% at 3 h post injection (p.i.) in healthy mice. The Kd value of 125I-anti-CD147 to CD147 protein was 6.344 nM, while that of 125I-IgG was over 100 nM. 125I-anti-CD147 showed much greater uptake in CD147 high-expression cancer cells compared to CD147 low-expression cancer cells. After intravenous injection in healthy mice, 125I-anti-CD147 showed high initial uptake in blood pool and liver, the uptake was decreased with time. The biological half-life of distribution and clearance phases in healthy mice were 0.63 h and 19.60 h, respectively. The effective dose of 124I-anti-CD147 was estimated as 0.104 mSv/MBq. We conducted immunoPET imaging in tumor-bearing mice, and demonstrated a significantly higher tumor-to-muscle ratio of 124I-anti-CD147 compared to that of 124I-IgG and 18F-FDG in CD147 (+) tumors. The expression levels of CD147 in cells and tumors were positively correlated with the maximum standardized uptake value (SUVmax) (P < 0.01). In conclusion, 124/125I-anti-CD147 displays high affinity to CD147, and represents potential for the imaging of CD147-positive tumors. The development of 124I-anti-CD147 may provide new insights into the regulation of tumor microenvironment and formulation of precision diagnosis and treatment programs for tumors.
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Fluordesoxiglucose F18 , Neoplasias da Próstata , Masculino , Humanos , Camundongos , Animais , Distribuição Tecidual , Compostos Radiofarmacêuticos , Radioisótopos do Iodo , Imunoglobulina G , Linhagem Celular Tumoral , Microambiente TumoralRESUMO
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.
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Desferroxamina , Ferro , Tomografia por Emissão de Pósitrons , Radioisótopos , Zircônio , Zircônio/química , Desferroxamina/química , Radioisótopos/química , Ferro/química , Tomografia por Emissão de Pósitrons/métodos , Piridonas/química , Deferiprona/química , Imunoconjugados/química , Compostos Radiofarmacêuticos/química , Anticorpos Monoclonais/químicaRESUMO
Invasive fungal infections have become a major challenge for public health, mainly due to the growing numbers of immunocompromised patients, with high morbidity and mortality. Currently, conventional imaging modalities such as computed tomography and magnetic resonance imaging contribute largely to the noninvasive diagnosis and treatment evaluation of those infections. These techniques, however, often fall short when a fast, noninvasive and specific diagnosis of fungal infection is necessary. Molecular imaging, especially using nuclear medicine-based techniques, aims to develop fungal-specific radiotracers that can be tested in preclinical models and eventually translated to human applications. In the last few decades, multiple radioligands have been developed and tested as potential fungal-specific tracers. These include radiolabeled peptides, antifungal drugs, siderophores, fungal-specific antibodies, and sugars. In this review, we provide an overview of the pros and cons of the available radiotracers. We also address the future prospects of fungal-specific imaging.
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Infecções Fúngicas Invasivas , Micoses , Humanos , Tomografia por Emissão de Pósitrons/métodos , Micoses/diagnóstico por imagem , Antifúngicos/uso terapêutico , Tomografia Computadorizada por Raios X , Anticorpos AntifúngicosRESUMO
This study aimed to establish the radio-immune imaging protocol on the basis of Avidin/Biotin system. The programmed death-ligand 1 (PD-L1) antibody (Atezolizumab) was employed as the primary molecule in targeting PD-L1, and the two-step strategy, consisting of the first injection of Avidin-conjugated PD-L1 monoclonal antibody (Atezolizumab) and the second injection of 7.4 MBq 68Ga-Biotin with a 60 h interval, was then verified on the colon cancer-bearing mice. PET imaging was performed at 30, 90, 180 min to measure the standard uptake value and tumor to liver ratios. Cellular binding experiments and in vivo distribution showed that the conjugation of Avidin did not affect the affinity of Atezolizumab to PD-L1 antigen. Biotin was radio-labeled with 68Ga with radiolabeling efficiency of 70.5 ± 3.5% and purification was needed to increase the radiochemical purity. For PD-L1-positive tumors, SUVmax was 0.38 ± 0.06 in the Avidin-Atezolizumab pre-treated mice at 90 min; the tumor/liver ratios of pre-targeting group were 1.06 ± 0.19 and 0.97 ± 0.16 at 30 and 90 min, while the absence of pre-treatment of Avidin was of the lower ratios as 0.88 ± 0.01 and 0.54 ± 0.11 when 68Ga-Biotin served as the radiopharmaceutical as well. In conclusion, pre-targeting immunoPET strategy can elevate the target-to-nontarget ratio, decrease the blood background and shorten the interval between injection of radiopharmaceuticals and PET scan, providing a highly PD-L1-specific and sensitive imaging method for the detection of tumorous immune micro-environment.
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Biotina , Neoplasias do Colo , Camundongos , Animais , Avidina , Antígeno B7-H1/metabolismo , Radioisótopos de Gálio , Tomografia por Emissão de Pósitrons/métodos , Compostos Radiofarmacêuticos , Linhagem Celular Tumoral , Microambiente TumoralRESUMO
PURPOSE: Cancer immunotherapies (CITs) have revolutionized the treatment of certain cancers, but many patients fail to respond or relapse from current therapies, prompting the need for new CIT agents. CD8+ T cells play a central role in the activity of many CITs, and thus, the rapid imaging of CD8+ cells could provide a critical biomarker for new CIT agents. However, existing 89Zr-labeled CD8 PET imaging reagents exhibit a long circulatory half-life and high radiation burden that limit potential applications such as same-day and longitudinal imaging. METHODS: To this end, we discovered and developed a 13-kDa single-domain antibody (VHH5v2) against human CD8 to enable high-quality, same-day imaging with a reduced radiation burden. To enable sensitive and rapid imaging, we employed a site-specific conjugation strategy to introduce an 18F radiolabel to the VHH. RESULTS: The anti-CD8 VHH, VHH5v2, demonstrated binding to a membrane distal epitope of human CD8 with a binding affinity (KD) of 500 pM. Subsequent imaging experiments in several xenografts that express varying levels of CD8 demonstrated rapid tumor uptake and fast clearance from the blood. High-quality images were obtained within 1 h post-injection and could quantitatively differentiate the tumor models based on CD8 expression level. CONCLUSION: Our work reveals the potential of this anti-human CD8 VHH [18F]F-VHH5v2 to enable rapid and specific imaging of CD8+ cells in the clinic.
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Neoplasias , Anticorpos de Domínio Único , Humanos , Linfócitos T CD8-Positivos , Tomografia por Emissão de Pósitrons/métodos , Neoplasias/diagnóstico por imagem , Linhagem Celular TumoralRESUMO
PURPOSE: Monoclonal antibody (mAb)-based PET (immunoPET) imaging can characterise tumour lesions non-invasively. It may be a valuable tool to determine which patients may benefit from treatment with a specific monoclonal antibody (mAb) and evaluate treatment response. For 89Zr immunoPET imaging, higher sensitivity of state-of-the art PET/CT systems equipped with silicon photomultiplier (SiPM)-based detector elements may be beneficial as the low positron abundance of 89Zr causes a low signal-to-noise level. Moreover, the long physical half-life limits the amount of activity that can be administered to the patients leading to poor image quality even when using long scan durations. Here, we investigated the difference in semiquantitative performance between the PMT-based Biograph mCT, our clinical reference system, and the SiPM-based Biograph Vision PET/CT in 89Zr immunoPET imaging. Furthermore, the effects of scan duration reduction using the Vision on semiquantitative imaging parameters and its influence on image quality assessment were evaluated. METHODS: Data were acquired on day 4 post 37 MBq 89Zr-labelled mAb injection. Five patients underwent a double scan protocol on both systems. Ten patients were scanned only on the Vision. For PET image reconstruction, three protocols were used, i.e. one camera-dependent protocol and European Association of Nuclear Medicine Research Limited (EARL) standards 1 and 2 compliant protocols. Vision data were acquired in listmode and were reprocessed to obtain images at shorter scan durations. Semiquantitative PET image parameters were derived from tumour lesions and healthy tissues to assess differences between systems and scan durations. Differently reconstructed images obtained using the Vision were visually scored regarding image quality by two nuclear medicine physicians. RESULTS: When images were reconstructed using 100% acquisition time on both systems following EARL standard 1 compliant reconstruction protocols, results regarding semiquantification were comparable. For Vision data, reconstructed images that conform to EARL1 standards still resulted in comparable semiquantification at shorter scan durations (75% and 50%) regarding 100% acquisition time. CONCLUSION: Scan duration of 89Zr immunoPET imaging using the Vision can be decreased up to 50% compared with using the mCT while maintaining image quality using the EARL1 compliant reconstruction protocol.
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Neoplasias , Tomografia por Emissão de Pósitrons combinada à Tomografia Computadorizada , Humanos , Tomografia por Emissão de Pósitrons combinada à Tomografia Computadorizada/métodos , Neoplasias/diagnóstico por imagem , Padrões de Referência , Anticorpos Monoclonais , Tomografia por Emissão de Pósitrons/métodos , Processamento de Imagem Assistida por ComputadorRESUMO
BACKGROUND: ZED8 is a novel monovalent antibody labeled with zirconium-89 for the molecular imaging of CD8. This work describes nonclinical studies performed in part to provide rationale for and to inform expectations in the early clinical development of ZED8, such as in the studies outlined in clinical trial registry NCT04029181 [1]. METHODS: Surface plasmon resonance, X-ray crystallography, and flow cytometry were used to characterize the ZED8-CD8 binding interaction, its specificity, and its impact on T cell function. Immuno-PET with ZED8 was assessed in huCD8+ tumor-bearing mice and in non-human primates. Plasma antibody levels were measured by ELISA to determine pharmacokinetic parameters, and OLINDA 1.0 was used to estimate radiation dosimetry from image-derived biodistribution data. RESULTS: ZED8 selectively binds to human CD8α at a binding site approximately 9 Å from that of MHCI making mutual interference unlikely. The equilibrium dissociation constant (KD) is 5 nM. ZED8 binds to cynomolgus CD8 with reduced affinity (66 nM) but it has no measurable affinity for rat or mouse CD8. In a series of lymphoma xenografts, ZED8 imaging was able to identify different CD8 levels concordant with flow cytometry. In cynomolgus monkeys with tool compound 89Zr-aCD8v17, lymph nodes were conspicuous by imaging 24 h post-injection, and the pharmacokinetics suggested a flat-fixed first-in-human dose of 4 mg per subject. The whole-body effective dose for an adult human was estimated to be 0.48 mSv/MBq, comparable to existing 89Zr immuno-PET reagents. CONCLUSION: 89Zr immuno-PET with ZED8 appears to be a promising biomarker of tissue CD8 levels suitable for clinical evaluation in cancer patients eligible for immunotherapy.
Assuntos
Neoplasias , Tomografia por Emissão de Pósitrons , Adulto , Humanos , Camundongos , Ratos , Animais , Tomografia por Emissão de Pósitrons/métodos , Indicadores e Reagentes/uso terapêutico , Distribuição Tecidual , Neoplasias/terapia , Neoplasias/tratamento farmacológico , Imunoterapia/métodos , Zircônio/química , Linfócitos T CD8-Positivos/metabolismo , Linhagem Celular TumoralRESUMO
PURPOSE: The recent conditional FDA approval of Aducanumab (Adu) for treating Alzheimer's disease (AD) and the continued discussions around that decision have increased interest in immunotherapy for AD and other brain diseases. Reliable techniques for brain imaging of antibodies may guide decision-making in the future but needs further development. In this study, we used 89Zr-immuno-PET to evaluate the targeting and distribution of a bispecific brain-shuttle IgG based on Adu with transferrin receptor protein-1 (TfR1) shuttling mechanism, mAbAdu-scFab8D3, designated Adu-8D3, as a candidate theranostic for AD. We also validated the 89Zr-immuno-PET platform as an enabling technology for developing new antibody-based theranostics for brain disorders. METHODS: Adu, Adu-8D3, and the non-binding control construct B12-8D3 were modified with DFO*-NCS and radiolabeled with 89Zr. APP/PS1 mice were injected with 89Zr-labeled mAbs and imaged on days 3 and 7 by positron emission tomography (PET). Ex vivo biodistribution was performed on day 7, and ex vivo autoradiography and immunofluorescence staining were done on brain tissue to validate the PET imaging results and target engagement with amyloid-ß plaques. Additionally, [89Zr]Zr-DFO*-Adu-8D3 was evaluated in 3, 7, and 10-month-old APP/PS1 mice to test its potential in early stage disease. RESULTS: A 7-fold higher brain uptake was observed for [89Zr]Zr-DFO*-Adu-8D3 compared to [89Zr]Zr-DFO*-Adu and a 2.7-fold higher uptake compared to [89Zr]Zr-DFO*-B12-8D3 on day 7. Autoradiography and immunofluorescence of [89Zr]Zr-DFO*-Adu-8D3 showed co-localization with amyloid plaques, which was not the case with the Adu and B12-8D3 conjugates. [89Zr]Zr-DFO*-Adu-8D3 was able to detect low plaque load in 3-month-old APP/PS1 mice. CONCLUSION: 89Zr-DFO*-immuno-PET revealed high and specific uptake of the bispecific Adu-8D3 in the brain and can be used for the early detection of Aß plaque pathology. Here, we demonstrate that 89Zr-DFO*-immuno-PET can be used to visualize and quantify brain uptake of mAbs and contribute to the evaluation of biological therapeutics for brain diseases.
Assuntos
Doença de Alzheimer , Radioisótopos , Camundongos , Animais , Distribuição Tecidual , Tomografia por Emissão de Pósitrons/métodos , Anticorpos Monoclonais , Doença de Alzheimer/diagnóstico por imagem , Doença de Alzheimer/terapia , Amiloide , Zircônio , Linhagem Celular TumoralRESUMO
Pancreatic ductal adenocarcinoma (PDAC) is a malignant tumor with insidious onset, rapid progression, and a very poor prognosis. CD47 is a transmembrane protein associated with the development and poor prognosis of pancreatic cancer. The aim of this study was to evaluate the diagnostic value of novel immunoPET tracers targeting CD47 in preclinical pancreatic cancer models. The association of CD47 expression with pancreatic cancer was analyzed using the Gene Expression Profiling Interactive Analysis platform. Immunohistochemical analysis of tissue microarrays was performed to detect CD47 expression in PDAC. CD47 expression levels on BxPC-3 and AsPC-1 cell membranes were compared using flow cytometry. A VHH (C2)-targeting human CD47 and its albumin-binding derivative (ABDC2) were labeled with 68Ga or 89Zr, respectively. The developed tracers were evaluated by immuno-positron emission tomography (immunoPET) imaging in tumor-bearing nude and CD47-humanized mice. [68Ga]Ga-NOTA-C2 effectively detected tumor lesions in nude mice models and further showed confirmative imaging capacity in CD47-humanized PDAC models. Compared with [68Ga]Ga-NOTA-C2, [89Zr]Zr-DFO-ABDC2 had a significantly prolonged circulation time, increased tumor uptake, and reduced accumulation in the kidneys. Finally, biodistribution and histological staining confirmed the results of the immunoPET imaging studies. In this study, we validated that two novel VHH-derived molecular imaging tracers for immunoPET imaging ([68Ga]Ga-NOTA-C2 and [89Zr]Zr-DFO-ABDC2) can effectively annotate CD47 expression and diagnose PDAC in a target-specific manner. Clinical application of the imaging strategies may help select patients for CD47-targeted therapies and assess the response thereafter.
Assuntos
Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Humanos , Animais , Camundongos , Radioisótopos de Gálio , Camundongos Nus , Distribuição Tecidual , Antígeno CD47 , Tomografia por Emissão de Pósitrons/métodos , Neoplasias Pancreáticas/diagnóstico por imagem , Neoplasias Pancreáticas/metabolismo , Carcinoma Ductal Pancreático/diagnóstico por imagem , Linhagem Celular Tumoral , Zircônio/química , Neoplasias PancreáticasRESUMO
Galectin-3 binding protein (Gal-3BP) is a glycoprotein that is overexpressed and secreted by several cancers and has been implicated as a marker of both tumor progression and poor prognosis in melanoma, non-small cell lung cancer, head and neck squamous cell carcinoma, and breast cancer. The expression of Gal-3BP by a variety of neoplasms makes it an enticing target for both diagnostics and therapeutics, including immuno-positron emission tomography (immunoPET) probes and antibody-drug conjugates (ADCs). Herein, we report the development, in vitro characterization, and in vivo evaluation of a pair of Gal-3BP-targeting radioimmunoconjugates for 89Zr-immunoPET. A humanized anti-Gal-3BP antibody, 1959, and its corresponding ADC, 1959-sss/DM4 (DM4 = ravtansine), were modified with desferrioxamine (DFO) to yield DFO-1959 and DFO-1959-sss/DM4 immunoconjugates bearing 1-2 DFO/monoclonal antibody. Both DFO-modified immunoconjugates retained their affinity for Gal-3BP in enzyme-linked immunosorbent assay experiments. The chelator-bearing antibodies were radiolabeled with zirconium-89 (t1/2 ≈ 3.3 d) to produce radioimmunoconjugates â [89Zr]Zr-DFO-1959 and [89Zr]Zr-DFO-1959-sss/DM4 â with high specific activity (>444 MBq/mg, >12 mCi/mg) and stability (>80% intact after 168 h in human serum at 37 °C). In mice bearing subcutaneous Gal-3BP-secreting A375-MA1 xenografts, [89Zr]Zr-DFO-1959 clearly delineated tumor tissue, reaching a maximum tumoral activity concentration (54.8 ± 15.8%ID/g) and tumor-to-background contrast (tumor-to-blood = 8.0 ± 4.6) at 120 h post-injection. The administration of [89Zr]Zr-DFO-1959 to mice bearing subcutaneous Gal-3BP-expressing melanoma patient-derived xenografts produced similarly promising results. [89Zr]Zr-DFO-1959 and [89Zr]Zr-DFO-1959-sss/DM4 exhibited nearly identical pharmacokinetic profiles in the mice bearing A375-MA1 tumors, though the latter produced higher uptake in the spleen and kidneys. Both [89Zr]Zr-DFO-1959 and [89Zr]Zr-DFO-1959-sss/DM4 effectively visualized Gal-3BP-secreting tumors in murine models of melanoma. These results suggest that both probes could play a role in the clinical imaging of Gal-3BP-expressing malignancies, particularly as companion theranostics for the identification of patients likely to respond to Gal-3BP-targeted therapeutics such as 1959-sss/DM4.
Assuntos
Carcinoma Pulmonar de Células não Pequenas , Imunoconjugados , Neoplasias Pulmonares , Melanoma , Animais , Humanos , Camundongos , Linhagem Celular Tumoral , Desferroxamina/química , Galectina 3 , Imunoconjugados/química , Tomografia por Emissão de Pósitrons/métodos , Zircônio/químicaRESUMO
PURPOSE: Pancreatic cancer is a malignant tumor with a high degree of malignancy, strong heterogeneity, and high lethality. Trop2 is a transmembrane glycoprotein associated with the occurrence, development, and poor prognosis of pancreatic cancer. This study aims to develop 64Cu/177Lu-labeled anti-Trop2 monoclonal antibody (hIMB1636) for positron emission tomography (PET) imaging and radioimmunotherapy (RIT) application in pancreatic cancer tumor models. METHODS: The binding kinetics of hIMB1636 to Trop2 antigen was measured by Biolayer interferometry (BLI). Western blotting was used to screen the Trop2 expression of pancreatic cancer cell lines. Flow cytometry and cell immunofluorescence were used to evaluate the binding ability of hIMB1636 and Trop2 on the cell surface. hIMB1636 were conjugated with p-SCN-Bn-NOTA (NOTA) and DOTA-NHS-ester (DOTA) for 64Cu and 177Lu radiolabeling respectively. ImmunoPET imaging and RIT studies were performed using 64Cu-NOTA-hIMB1636 and 177Lu-DOTA-hIMB1636 in subcutaneous pancreatic cancer tumor models. RESULTS: hIMB1636 had a strong binding affinity to Trop2 according to the results of BLI. The T3M-4 cell line showed the strongest expression of Trop2 and specific binding ability of hIMB1636 according to the results of Western blotting, flow cytometry, and cell immunofluorescence. The radiochemical purity of 64Cu-NOTA-hIMB1636 and 177Lu-DOTA-hIMB1636 exceeded 95%. PET imaging showed gradually an accumulation of 64Cu-NOTA-hIMB1636 in T3M-4 tumor models. The maximum tumor uptake was 8.95 ± 1.07%ID/g (n = 4) at 48 h post injection (p.i.), which had significant differences with T3M-4-blocked and PaTu8988-negative groups (P < 0.001). The high-177Lu-hIMB1636 group demonstrated the strongest tumor suppression with standardized tumor volume about 94.24 ± 14.62% (n = 5) at 14 days p.i., significantly smaller than other groups (P < 0.05). Ex vivo biodistribution and histological staining verified the in vivo PET imaging and RIT results. CONCLUSIONS: This study demonstrated that 64Cu/177Lu-labeled hIMB1636 could noninvasively evaluate the expression level of Trop2 and inhibit the Trop2-overexpressed tumor growth in pancreatic cancer tumor models. Further clinical evaluation and translation of Trop2-targeted drug may be of great help in the stratification and management of pancreatic cancer patients.
Assuntos
Neoplasias Pancreáticas , Medicina de Precisão , Humanos , Distribuição Tecidual , Linhagem Celular Tumoral , Tomografia por Emissão de Pósitrons/métodos , Neoplasias Pancreáticas/diagnóstico por imagem , Neoplasias Pancreáticas/radioterapia , Anticorpos Monoclonais/uso terapêutico , Anticorpos Monoclonais/metabolismo , Neoplasias PancreáticasRESUMO
PURPOSE: Abnormal CD38 expression in some hematologic malignancies, including lymphoma, has made it a biomarker for targeted therapies. Daratumumab (Dara) is the first FDA-approved CD38-specific monoclonal antibody, enabling successfully immunoPET imaging over the past years. Radiolabeled Dara however has a long blood circulation and delayed tumor uptake which can limit its applications. The focus of this study is to develop 64Cu-labeled Dara-F(ab')2 for the visualization of CD38 in lymphoma models. METHODS: F(ab')2 fragment was prepared from Dara using an IdeS enzyme and purified with Protein A beads. Western blotting, flow cytometry, and surface plasmon resonance (SPR) were performed for in vitro assay. Probes were labeled with 64Cu after the chelation of 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA). Small animal PET imaging and quantitative analysis were performed after injection of 64Cu-labeled Dara-F(ab')2, IgG-F(ab')2, and Dara for evaluation in lymphoma models. RESULTS: Flow cytometry and SPR assay proved the specific binding ability of Dara-F(ab')2 and NOTA-Dara-F(ab')2 in vitro. Radiolabeling yield of [64Cu]Cu-NOTA-Dara-F(ab')2 was over 90% and with a specific activity of 4.0 ± 0.6 × 103 MBq/µmol (n = 5). PET imaging showed [64Cu]Cu-NOTA-Dara-F(ab')2 had a rapid and high tumor uptake as early as 2 h (6.9 ± 1.2%ID/g) and peaked (9.5 ± 0.7%ID/g) at 12 h, whereas [64Cu]Cu-NOTA-Dara reached its tumor uptake peaked at 48 h (8.3 ± 1.4%ID/g, n = 4). In comparison, IgG-F(ab')2 and HBL-1 control groups found no noticeable tumor uptake. [64Cu]Cu-NOTA-Dara-F(ab')2 had significantly lower uptake in blood pool, bone, and muscle than [64Cu]Cu-NOTA-Dara and its tumor-to-blood and tumor-to-muscle ratios were significantly higher than controls. CONCLUSIONS: [64Cu]Cu-NOTA-Dara-F(ab')2 showed a rapid and high tumor uptake in CD38-positive lymphoma models with favorable imaging contrast, showing its promise as a potential PET imaging agent for future clinical applications.
Assuntos
Anticorpos Monoclonais , Linfoma , Animais , Linhagem Celular Tumoral , Humanos , Fragmentos Fab das Imunoglobulinas/metabolismo , Imunoglobulina G , Linfoma/diagnóstico por imagem , Tomografia por Emissão de Pósitrons/métodosRESUMO
PURPOSE: Early detection of hepatocellular carcinoma (HCC) remains a clinical challenge. Glypican 3 (GPC3) is a proteoglycan highly specific for HCC and is a potential diagnostic and therapeutic target for HCC. This work aims to develop GPC3-targeted immuno-positron emission tomography (immunoPET) imaging strategies and to assess the diagnostic values in preclinical HCC models. METHODS: Flow cytometry was used to screen GPC3-positive HCC cell lines. The expression of GPC3 in HCCs was detected by immunohistochemistry on tissue microarray. A novel GPC3-specific single domain antibody (sdAb) was produced and labeled with gallium-68 (68Ga, T1/2 = 1.1 h) and fluorine-18 (18F, T1/2 = 1.8 h) to develop radiotracers with different half-lives. The diagnostic efficacies of the developed probes (i.e., [68Ga]Ga-NOTA-G2, [18F]F-G2, and [68Ga]Ga-NOTA-ABDG2) were interrogated in preclinical HCC models bearing varying GPC3 levels. RESULTS: GPC3 was strongly expressed on HCC cell lines and patients with poorly differentiated HCC. [68Ga]Ga-NOTA-G2 immunoPET imaging specifically delineated the subcutaneous HCC lesions, outperforming the traditional 18F-fluorodeoxyglucose PET and the nonspecific [68Ga]Ga-NOTA-NbGFP immunoPET. ImmunoPET imaging with [18F]F-G2 also efficiently diagnosed the tumors with clarity. Moreover, the fusion of G2 to an albumin-binding domain (ABD) significantly increased the tumor uptake and decreased kidney accumulation of the radiotracer when compared to [68Ga]Ga-NOTA-G2. CONCLUSIONS: In the work, we successfully developed sdAb-derived GPC3-targeted immunoPET imaging strategies and characterized the superior diagnostic accuracies in preclinical HCC models. Furthermore, we synthesized a fusion protein ABDG2 with improved targeting and pharmacokinetic properties, serving as a promising candidate for developing radioimmunotherapy agents.