RESUMO
PURPOSE: Immune checkpoint inhibitors (ICI) targeting the programmed cell death protein 1 and its ligand (PD-1/PD-L1) have transformed the treatment paradigm for metastatic renal cell carcinoma (RCC). However, response rates to ICIs as single agents or in combination vary widely and predictive biomarkers are lacking. Possibly related to the heterogeneity and dynamic nature of PD-L1 expression, tissue-based methods have shown limited value. Immuno-positron emission tomography (immunoPET) may enable noninvasive, comprehensive, and real-time PD-L1 detection. Herein, we systematically examined the performance of immunoPET for PD-L1 detection relative to IHC in an RCC patient-derived tumorgraft (TG) platform. EXPERIMENTAL DESIGN: Eight independent RCC TGs with a wide range of PD-L1 expression (0%-85%) were evaluated by immunoPET. Uptake of 89Zr-labeled atezolizumab ([89Zr]Zr-DFO-ATZ) was compared with PD-L1 expression in tumors by IHC through double-blind analyses. Clinical outcomes of ICI-treated patients whose TGs were examined were analyzed to evaluate the clinical role of immunoPET in RCC. RESULTS: ImmunoPET with [89Zr]Zr-DFO-ATZ (day 6/7 postinjection) revealed a statistically significant association with PD-L1 IHC assays (P = 0.0014; correlation ρXY = 0.78). Furthermore, immunoPET can be used to assess the heterogeneous distribution of PD-L1 expression. Finally, studies in the corresponding patients (n = 4) suggest that PD-L1 signal may influence ICI responsiveness. CONCLUSIONS: ImmunoPET with [89Zr]Zr-DFO-ATZ may enable a thorough and dynamic assessment of PD-L1 across sites of disease. The power of immunoPET to predict ICI response in RCC is being explored in an ongoing clinical trial (NCT04006522).
Assuntos
Carcinoma de Células Renais , Neoplasias Renais , Humanos , Antígeno B7-H1/metabolismo , Carcinoma de Células Renais/diagnóstico por imagem , Carcinoma de Células Renais/tratamento farmacológico , Neoplasias Renais/diagnóstico por imagem , Neoplasias Renais/tratamento farmacológico , Radioisótopos , Distribuição Tecidual , Zircônio , Ensaios Clínicos como AssuntoRESUMO
We previously reported the design and synthesis of a small-molecule drug conjugate (SMDC) platform that demonstrated several advantages over antibody-drug conjugates (ADCs) in terms of in vivo pharmacokinetics, solid tumor penetration, definitive chemical structure, and adaptability for modular synthesis. Constructed on a tri-modal SMDC platform derived from 1,3,5-triazine (TZ) that consists of a targeting moiety (Lys-Urea-Glu) for prostate-specific membrane antigen (PSMA), here we report a novel class of chemically identical theranostic small-molecule prodrug conjugates (T-SMPDCs), [18/19F]F-TZ(PSMA)-LEGU-TLR7, for PSMA-targeted delivery and controlled release of toll-like receptor 7 (TLR7) agonists to elicit de novo immune response for cancer immunotherapy. In vitro competitive binding assay of [19F]F-TZ(PSMA)-LEGU-TLR7 showed that the chemical modification of Lys-Urea-Glu did not compromise its binding affinity to PSMA. Receptor-mediated cell internalization upon the PSMA binding of [18F]F-TZ(PSMA)-LEGU-TLR7 showed a time-dependent increase, indicative of targeted intracellular delivery of the theranostic prodrug conjugate. The designed controlled release of gardiquimod, a TLR7 agonist, was realized by a legumain cleavable linker. We further performed an in vivo PET/CT imaging study that showed significantly higher uptake of [18F]F-TZ(PSMA)-LEGU-TLR7 in PSMA+ PC3-PIP tumors (1.9 ± 0.4% ID/g) than in PSMA- PC3-Flu tumors (0.8 ± 0.3% ID/g) at 1 h post-injection. In addition, the conjugate showed a one-compartment kinetic profile and in vivo stability. Taken together, our proof-of-concept biological evaluation demonstrated the potential of our T-SMPDCs for cancer immunomodulatory therapies.
Assuntos
Pró-Fármacos , Neoplasias da Próstata , Antígenos de Superfície/metabolismo , Linhagem Celular Tumoral , Preparações de Ação Retardada , Glutamato Carboxipeptidase II/metabolismo , Humanos , Masculino , Tomografia por Emissão de Pósitrons combinada à Tomografia Computadorizada/métodos , Medicina de Precisão , Pró-Fármacos/farmacologia , Neoplasias da Próstata/metabolismo , Receptor 7 Toll-Like , UreiaRESUMO
In the past two decades, extensive efforts have been made to develop agents targeting prostate-specific membrane antigen (PSMA) for prostate cancer imaging and therapy. To date, represented by two recent approvals of [68Ga]Ga-PSMA-11 and [18F]F-DCFPyL by the United States Food and Drug Administration (US-FDA) for positron emission tomography (PET) imaging to identify suspected metastases or recurrence in patients with prostate cancer, PSMA-targeting imaging and theranostic agents derived from small molecule PSMA inhibitors have advanced to clinical practice and trials of prostate cancer. The focus of current development of new PSMA-targeting agents has thus shifted to the improvement of in vivo pharmacokinetics and higher specific binding affinity with the aims to further increase the detection sensitivity and specificity and minimize the toxicity to non-target tissues, particularly the kidneys. The main strategies involve systematic chemical modifications of the linkage between the targeting moiety and imaging/therapy payloads. In addition to a summary of the development history of PSMA-targeting agents, this review provides an overview of current advances and future promise of PSMA-targeted imaging and theranostics with focuses on the structural determinants of the chemical modification towards the next generation of PSMA-targeting agents.
Assuntos
Antígenos de Superfície/metabolismo , Glutamato Carboxipeptidase II/antagonistas & inibidores , Glutamato Carboxipeptidase II/metabolismo , Tomografia por Emissão de Pósitrons/métodos , Neoplasias da Próstata/diagnóstico por imagem , Neoplasias da Próstata/radioterapia , Compostos Radiofarmacêuticos/uso terapêutico , Humanos , Masculino , Medicina de Precisão , Neoplasias da Próstata/patologia , Compostos Radiofarmacêuticos/metabolismoRESUMO
Neuroendocrine prostate cancer (NEPC) is an aggressive and lethal variant of prostate cancer (PCa), and it remains a diagnostic challenge. Herein we report our findings of using synaptic vesicle glycoprotein 2 isoform A (SV2A) as a promising marker for positron emission tomography (PET) imaging of neuroendocrine differentiation (NED). The bioinformatic analyses revealed an amplified SV2A gene expression in clinical samples of NEPC versus castration-resistant PCa with adenocarcinoma characteristics (CRPC-Adeno). Importantly, significantly upregulated SV2A protein levels were found in both NEPC cell lines and tumor tissues. PET imaging studies were carried out in NEPC xenograft models with 18F-SynVesT-1. Although 18F-SynVesT-1 is not a cancer imaging agent, it showed a significant uptake level in the SV2A+ tumor (NCI-H660: 0.70 ± 0.14 %ID/g at 50-60 min p.i.). The SV2A blockade resulted in a significant reduction of tumor uptake (0.25 ± 0.03 %ID/g, p = 0.025), indicating the desired SV2A imaging specificity. Moreover, the comparative PET imaging study showed that the DU145 tumors could be clearly visualized by 18F-SynVesT-1 but not 68Ga-PSMA-11 nor 68Ga-DOTATATE, further validating the role of SV2A-targeted imaging for noninvasive assessment of NED in PCa. In conclusion, we demonstrated that SV2A, highly expressed in NEPC, can serve as a promising target for noninvasive imaging evaluation of NED.
Assuntos
Carcinoma Neuroendócrino/diagnóstico por imagem , Glicoproteínas de Membrana/análise , Proteínas do Tecido Nervoso/análise , Tomografia por Emissão de Pósitrons/métodos , Neoplasias da Próstata/diagnóstico por imagem , Animais , Carcinoma Neuroendócrino/metabolismo , Linhagem Celular Tumoral , Humanos , Masculino , Camundongos , Compostos Organometálicos , Neoplasias da Próstata/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The prolonged in vivo persistence of antibodies results in high background and poor contrast during their use as molecular imaging agents for positron emission tomography (PET). We have recently described a class of engineered Fc fusion proteins that selectively deplete antigen-specific antibodies without affecting the levels of antibodies of other specificities. Here, we demonstrate that these Fc fusions (called Seldegs, for selective degradation) can be used to clear circulating, radiolabeled HER2-specific antibody during diagnostic imaging of HER2-positive tumors in mice. The analyses show that Seldegs have considerable promise for the reduction of whole-body exposure to radiolabel and improvement of contrast during PET.
Assuntos
Neoplasias , Tomografia por Emissão de Pósitrons , Animais , Anticorpos , Linhagem Celular Tumoral , Camundongos , Tomografia por Emissão de Pósitrons/métodos , Receptor ErbB-2RESUMO
Copper-67 (t1/2 = 2.58 days) decays by ß- ([Formula: see text]: 562 keV) and γ-rays (93 keV and 185 keV) rendering it with potential for both radionuclide therapy and single-photon emission computed tomography (SPECT) imaging. Prompted by the recent breakthrough of 67Cu production with high specific activity, high radionuclidic purity, and sufficient quantities, the interest in the theranostic potential of 67Cu has been rekindled. This work addresses the practicability of developing 67Cu-labeled antibodies with substantially improved quality for cancer radioimmunotheranostics. Proof of concept is demonstrated with pertuzumab, a US-FDA-approved monoclonal antibody for combination therapies of HER2-positive breast cancer. With an average number of 1.9 chelators coupled to each antibody, we achieved a two-order of magnitude increase in radiolabeling efficiency compared to literature reports. In a preclinical therapeutic study, mice (n = 4-7/group) bearing HER2+ xenografts exhibited a 67Cu-dose dependent tumor-growth inhibition from 67Cu-labeled-Pertuzumab co-administered with trastuzumab. Furthermore, greater tumor size reduction was observed with 67Cu-labeled-pertuzumab formulations of higher specific activity. The potential of SPECT imaging with 67Cu radiopharmaceuticals was tested after 67Cu-labeled-Pertuzumab administration. Impressively, all tumors were clearly visualized by SPECT imaging with 67Cu-labeled-Pertuzumab even at day 5 post injection. This work demonstrates it is practical to use 67Cu radioimmunoconjugates for cancer radioimmunotheranostics.
Assuntos
Radioisótopos de Cobre/uso terapêutico , Imunoconjugados/uso terapêutico , Imunoterapia , Tomografia Computadorizada de Emissão de Fóton Único , Animais , Anticorpos Monoclonais Humanizados , Linhagem Celular Tumoral , Feminino , Humanos , Camundongos SCID , Radioimunoterapia , Receptor ErbB-2/metabolismo , Tomografia Computadorizada por Raios X , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BACKGROUND: Malignant peripheral nerve sheath tumor (MPNST) is an aggressive form of soft-tissue sarcoma (STS) in children. Despite intensive therapy, relatively few children with metastatic and unresectable disease survive beyond three years. RAS pathway activation is common in MPNST, suggesting MEK pathway inhibition as a targeted therapy, but the impact on clinical outcome has been small to date. PROCEDURE: We conducted preclinical pharmacokinetic (PK) and pharmacodynamic studies of two MEK inhibitors, trametinib and selumetinib, in two MPNST models and analyzed tumors for intratumor drug levels. We then investigated 3'-deoxy-3'-[18 F]fluorothymidine (18 F-FLT) PET imaging followed by 18 F-FDG PET/CT imaging of MPNST xenografts coupled to short-term or longer-term treatment with selumetinib focusing on PET-based imaging as a biomarker of MEK inhibition. RESULTS: Trametinib decreased pERK expression in MPNST xenografts but did not prolong survival or decrease Ki67 expression. In contrast, selumetinib prolonged survival of animals bearing MPNST xenografts, and this correlated with decreased pERK and Ki67 staining. PK studies revealed a significantly higher fraction of unbound selumetinib within a responsive MPNST xenograft model. Thymidine uptake, assessed by 18 F-FLT PET/CT, positively correlated with Ki67 expression in different xenograft models and in response to selumetinib. CONCLUSION: The ability of MEK inhibitors to control MPNST growth cannot simply be predicted by serum drug levels or drug-induced changes in pERK expression. Tumor cell proliferation assessed by 18 F-FLT PET imaging might be useful as an early response marker to targeted therapies, including MEK inhibition, where a primary effect is cell-cycle arrest.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Neurofibrossarcoma/patologia , Tomografia por Emissão de Pósitrons combinada à Tomografia Computadorizada/métodos , Proteínas ras/antagonistas & inibidores , Animais , Apoptose , Benzimidazóis/administração & dosagem , Proliferação de Células , Fluordesoxiglucose F18/farmacocinética , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Neurofibrossarcoma/diagnóstico por imagem , Neurofibrossarcoma/tratamento farmacológico , Neurofibrossarcoma/metabolismo , Piridonas/administração & dosagem , Pirimidinonas/administração & dosagem , Compostos Radiofarmacêuticos/farmacocinética , Distribuição Tecidual , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Owing to the diversity of cancer types and the spatiotemporal heterogeneity of tumour signals, high-resolution imaging of occult malignancy is challenging. 18F-fluorodeoxyglucose positron emission tomography allows for near-universal cancer detection, yet in many clinical scenarios it is hampered by false positives. Here, we report a method for the amplification of imaging contrast in tumours via the temporal integration of the imaging signals triggered by tumour acidosis. This method exploits the catastrophic disassembly, at the acidic pH of the tumour milieu, of pH-sensitive positron-emitting neutral copolymer micelles into polycationic polymers, which are then internalized and retained by the cancer cells. Positron emission tomography imaging of the 64Cu-labelled polymers detected small occult tumours (10-20 mm3) in the brain, head, neck and breast of mice at much higher contrast than 18F-fluorodeoxyglucose, 11C-methionine and pH-insensitive 64Cu-labelled nanoparticles. We also show that the pH-sensitive probes reduce false positive detection rates in a mouse model of non-cancerous lipopolysaccharide-induced inflammation. This macromolecular strategy for integrating tumour acidosis should enable improved cancer detection, surveillance and staging.
Assuntos
Acidose/diagnóstico por imagem , Cobre/química , Neoplasias/diagnóstico por imagem , Polímeros/química , Tomografia por Emissão de Pósitrons/métodos , Coloração e Rotulagem/métodos , Animais , Neoplasias da Mama , Linhagem Celular Tumoral , Modelos Animais de Doenças , Feminino , Concentração de Íons de Hidrogênio , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Camundongos SCID , Nanopartículas/química , Nanotecnologia/métodos , Neoplasias/patologiaRESUMO
Various glutamate urea ligands have displayed high affinities to prostate specific membrane antigen (PSMA), which is highly overexpressed in prostate and other cancer sites. The multivalent versions of small PSMA-targeted molecules are known to be even more efficiently bound to the receptor. Here, we employ a well-known urea-based ligand, 2-[3-(1,3-dicarboxypropyl)-ureido] pentanedioic acid (DUPA) and triazine dendrimers in order to study the effect of molecular size on multivalent targeting in prostate cancer. The synthetic route starts with the preparation of a dichlorotriazine bearing DUPA in 67% overall yield over five steps. This dichlorotriazine reacts with G1, G3, and G5 triazine dendrimers bearing a 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) group for 64Cu-labeling at the core to afford poly(monochlorotriazine) intermediates. Addition of 4-aminomethylpiperidine (4-AMP) and the following deprotection produce the target compounds, G1-(DUPA)4, G3-(DUPA)16, and G5-(DUPA)64. These targets include 4/16/64 DUPA groups on the surface and a DOTA group at the core, respectively. In vitro cell assay using PC3-PIP (PSMA positive) and PC3-FLU (PSMA negative) cells reveals that G1-(DUPA)4 has the highest PC3-PIP to PC3-FLU uptake ratio (10-fold) through the PSMA-mediated specific uptake. While G5-(DUPA)64 displayed approximately 12 times higher binding affinity (IC50 23.6 nM) to PC3-PIP cells than G1-(DUPA)4 (IC50 282.3 nM) as evaluated in a competitive binding assay, the G5 dendrimer also showed high non-specific binding to PC3-FLU cells. In vivo uptake of the 64Cu-labeled dendrimers was also evaluated in severe combined inmmunodeficient (SCID) mice bearing PC3-PIP and PC3-FLU xenografts on each shoulder, respectively. Interestingly, quantitative imaging analysis of positron emission tomograph (PET) displayed the lowest tumor uptake in PC3-PIP cells for the midsize dendrimer G3-(DUPA)16 (19.4 kDa) (0.66 ± 0.15%ID/g at 1 h. p.i., 0.64 ± 0.11%ID/g at 4 h. p.i., and 0.67 ± 0.08%ID/g at 24 h. p.i.). Through the specific binding of G1-(DUPA)4 to PSMA, the smallest dendrimer (5.1 kDa) demonstrated the highest PC3-PIP to muscle and PC3-PIP to PC3-FLU uptake ratios (17.7 ± 5.5 and 6.7 ± 3.0 at 4 h p.i., respectively). In addition, the enhanced permeability and retention (EPR) effect appeared to be an overwhelming factor for tumor uptake of the largest dendrimer G5-(DUPA)64 as the uptake was at a similar level irrelevant to the PSMA expression.
Assuntos
Dendrímeros/química , Dendrímeros/farmacocinética , Glutamato Carboxipeptidase II/metabolismo , Glicoproteínas de Membrana/metabolismo , Neoplasias da Próstata/metabolismo , Triazinas/química , Animais , Transporte Biológico , Linhagem Celular Tumoral , Glutaratos/química , Humanos , Masculino , Camundongos , Tomografia por Emissão de Pósitrons , Neoplasias da Próstata/diagnóstico por imagem , Ureia/análogos & derivados , Ureia/químicaRESUMO
BACKGROUND: Programmed death-ligand 1 (PD-L1) expression in metastatic renal cell carcinoma (RCC) correlates with a worse prognosis, but whether it also predicts responsiveness to anti-PD-1/PD-L1 therapy remains unclear. Most studies of PD-L1 are limited by evaluation in primary rather than metastatic sites, and in biopsy samples, which may not be representative. These limitations may be overcome with immuno-positron emission tomography (iPET), an emerging tool allowing the detection of cell surface proteins with radiolabeled antibodies. Here, we report iPET studies of PD-L1 in a preclinical tumorgraft model of clear cell RCC (ccRCC) from a patient who had a favorable response to anti-PD-1 therapy. CASE PRESENTATION: A 49-year-old man underwent a cytoreductive nephrectomy in 2017 of a right kidney tumor invading into the adrenal gland that was metastatic to the lungs and a rib. Histological analyses revealed a ccRCC of ISUP grade 4 with extensive sarcomatoid features. IMDC risk group was poor. Within two hours of surgery, a tumor sample was implanted orthotopically into NOD/SCID mice. Consistent with an aggressive tumor, a renal mass was detected 18 days post-implantation. Histologically, the tumorgraft showed sarcomatoid differentiation and high levels of PD-L1, similar to the patient's tumor. PD-L1 was evaluated in subsequently transplanted mice using iPET and the results were compared to control mice implanted with a PD-L1-negative tumor. We labeled atezolizumab, an anti-PD-L1 antibody with a mutant Fc, with zirconium-89. iPET revealed significantly higher 89Zr-atezolizumab uptake in index than control tumorgrafts. The patient was treated with high-dose IL2 initially, and subsequently with pazopanib, with rapidly progressive disease, but had a durable response with nivolumab. CONCLUSIONS: To our knowledge, this is the first report of non-invasive detection of PD-L1 in renal cancer using molecular imaging. This study supports clinical evaluation of iPET to identify RCC patients with tumors deploying the PD-L1 checkpoint pathway who may be most likely to benefit from PD-1/PD-L1 disrupting drugs.
Assuntos
Anticorpos Monoclonais Humanizados , Antígeno B7-H1/metabolismo , Carcinoma de Células Renais/diagnóstico , Carcinoma de Células Renais/metabolismo , Tomografia por Emissão de Pósitrons , Radioisótopos , Compostos Radiofarmacêuticos , Zircônio , Animais , Antineoplásicos Imunológicos/administração & dosagem , Antineoplásicos Imunológicos/efeitos adversos , Antineoplásicos Imunológicos/uso terapêutico , Biomarcadores Tumorais , Carcinoma de Células Renais/tratamento farmacológico , Humanos , Imuno-Histoquímica , Masculino , Camundongos , Pessoa de Meia-Idade , Nivolumabe/administração & dosagem , Nivolumabe/efeitos adversos , Nivolumabe/uso terapêutico , Tomografia por Emissão de Pósitrons/métodos , Tomografia Computadorizada por Raios X , Resultado do TratamentoRESUMO
Hypoxia is regarded as a potential prognostic biomarker for tumor aggressiveness, progression, and response to therapy. The radiotracer 18F-fluoromisonidazole ([18F]FMISO) has been used with positron emission tomography (PET) to reveal tumor hypoxia. Meanwhile, blood oxygen level dependent (BOLD) MRI and tissue oxygen level dependent (TOLD) MRI offer insight into oxygenation based on endogenous signals without the need for radiolabels. Here, we compared BOLD and TOLD MRI with [18F]FMISO uptake using Dunning prostate R3327-AT1 tumor bearing rats. BOLD and TOLD MRI were acquired with respect to an oxygen gas breathing challenge. The following day, dynamic PET was performed up to 90 minutes following IV injection of [18F]FMISO. Tumors showed distinct heterogeneity based on each technique. Correlations were observed between magnitude of mean BOLD or TOLD MRI signal responses to oxygen-breathing challenge and initial distribution of [18F]FMISO. Correlations were observed for whole tumor as well on a regional basis with stronger correlations in the well perfused tumor periphery indicating the strong influence of perfused vasculature. After 90 minutes most correlations with signal intensity became quite weak, but correlations were observed between hypoxic fraction based on FMISO and fractions of tumor showing BOLD or TOLD response in a subset of tumors. This emphasizes the importance of considering regional heterogeneity and responsive fractions, as opposed to simple magnitudes of responses. Although the data represent a small cohort of tumors they present direct correlations between oxygen sensitive MRI and PET hypoxia reporter agents in the same tumors, indicating the potential utility of further investigations.
RESUMO
Programmed death-ligand 1 (PD-L1) expression on tumor cells is essential for T cell impairment, and PD-L1 blockade therapy has shown unprecedented durable responses in several clinical studies. Although higher expression of PD-L1 on tumor cells is associated with a better immune response after Ab blockade, some PD-L1-negative patients also respond to this therapy. In the current study, we explored whether PD-L1 on tumor or host cells was essential for anti-PD-L1-mediated therapy in 2 different murine tumor models. Using real-time imaging in whole tumor tissues, we found that anti-PD-L1 Ab accumulates in tumor tissues, regardless of the status of PD-L1 expression on tumor cells. We further observed that, while PD-L1 on tumor cells was largely dispensable for the response to checkpoint blockade, PD-L1 in host myeloid cells was essential for this response. Additionally, PD-L1 signaling in defined antigen-presenting cells (APCs) negatively regulated and inhibited T cell activation. PD-L1 blockade inside tumors was not sufficient to mediate regression, as limiting T cell trafficking reduced the efficacy of the blockade. Together, these findings demonstrate that PD-L1 expressed in APCs, rather than on tumor cells, plays an essential role in checkpoint blockade therapy, providing an insight into the mechanisms of this therapy.
Assuntos
Antígeno B7-H1/imunologia , Neoplasias/imunologia , Neoplasias/terapia , Animais , Anticorpos Monoclonais/imunologia , Células Apresentadoras de Antígenos/imunologia , Antígeno B7-H1/antagonistas & inibidores , Linhagem Celular , Linhagem Celular Tumoral , Citometria de Fluxo , Imunidade Celular , Imunoterapia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Células Mieloides/metabolismo , Microambiente Tumoral/imunologiaRESUMO
Clear cell renal cell carcinoma (ccRCC) is characterized by inactivation of the von Hippel-Lindau tumour suppressor gene (VHL). Because no other gene is mutated as frequently in ccRCC and VHL mutations are truncal, VHL inactivation is regarded as the governing event. VHL loss activates the HIF-2 transcription factor, and constitutive HIF-2 activity restores tumorigenesis in VHL-reconstituted ccRCC cells. HIF-2 has been implicated in angiogenesis and multiple other processes, but angiogenesis is the main target of drugs such as the tyrosine kinase inhibitor sunitinib. HIF-2 has been regarded as undruggable. Here we use a tumourgraft/patient-derived xenograft platform to evaluate PT2399, a selective HIF-2 antagonist that was identified using a structure-based design approach. PT2399 dissociated HIF-2 (an obligatory heterodimer of HIF-2α-HIF-1ß) in human ccRCC cells and suppressed tumorigenesis in 56% (10 out of 18) of such lines. PT2399 had greater activity than sunitinib, was active in sunitinib-progressing tumours, and was better tolerated. Unexpectedly, some VHL-mutant ccRCCs were resistant to PT2399. Resistance occurred despite HIF-2 dissociation in tumours and evidence of Hif-2 inhibition in the mouse, as determined by suppression of circulating erythropoietin, a HIF-2 target and possible pharmacodynamic marker. We identified a HIF-2-dependent gene signature in sensitive tumours. Gene expression was largely unaffected by PT2399 in resistant tumours, illustrating the specificity of the drug. Sensitive tumours exhibited a distinguishing gene expression signature and generally higher levels of HIF-2α. Prolonged PT2399 treatment led to resistance. We identified binding site and second site suppressor mutations in HIF-2α and HIF-1ß, respectively. Both mutations preserved HIF-2 dimers despite treatment with PT2399. Finally, an extensively pretreated patient whose tumour had given rise to a sensitive tumourgraft showed disease control for more than 11 months when treated with a close analogue of PT2399, PT2385. We validate HIF-2 as a target in ccRCC, show that some ccRCCs are HIF-2 independent, and set the stage for biomarker-driven clinical trials.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/antagonistas & inibidores , Carcinoma de Células Renais/tratamento farmacológico , Carcinoma de Células Renais/metabolismo , Indanos/farmacologia , Indanos/uso terapêutico , Neoplasias Renais/tratamento farmacológico , Neoplasias Renais/metabolismo , Sulfonas/farmacologia , Sulfonas/uso terapêutico , Animais , Translocador Nuclear Receptor Aril Hidrocarboneto/genética , Translocador Nuclear Receptor Aril Hidrocarboneto/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Sítios de Ligação , Carcinoma de Células Renais/genética , Carcinoma de Células Renais/patologia , Linhagem Celular Tumoral , Transformação Celular Neoplásica , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Eritropoetina/antagonistas & inibidores , Eritropoetina/sangue , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Indanos/administração & dosagem , Indóis/farmacologia , Indóis/uso terapêutico , Neoplasias Renais/genética , Neoplasias Renais/patologia , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Terapia de Alvo Molecular , Mutação , Pirróis/farmacologia , Pirróis/uso terapêutico , Reprodutibilidade dos Testes , Sulfonas/administração & dosagem , Sunitinibe , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Targeted therapy has become an effective strategy of precision medicine for cancer treatment. Based on the success of antibody-drug conjugates (ADCs), here we report a theranostic design of small-molecule drug conjugates (T-SMDCs) for targeted imaging and chemotherapy of prostate cancer. The structure of T-SMDCs built upon a polyethylene glycol (PEG) scaffold consists of (i) a chelating moiety for positron emission tomography (PET) imaging when labeled with (68)Ga, a positron-emitting radioisotope; (ii) a prostate specific membrane antigen (PSMA) specific ligand for prostate cancer targeting; and (iii) a cytotoxic drug (DM1) for chemotherapy. For proof-of-concept, such a T-SMDC, NO3A-DM1-Lys-Urea-Glu, was synthesized and evaluated. The chemical modification of Lys-Urea-Glu for the construction of the conjugate did not compromise its specific binding affinity to PSMA. The PSMA-mediated internalization of (68)Ga-labeled NO3A-DM1-Lys-Urea-Glu displayed a time-dependent manner, allowing the desired drug delivery and release within tumor cells. The antiproliferative activity of the T-SMDC showed a positive correlation with the PSMA expression level. Small animal PET imaging with (68)Ga-labeled NO3A-DM1-Lys-Urea-Glu exhibited significantly higher uptake (p < 0.01) in the PSMA positive PC3-PIP tumors (4.30 ± 0.20%ID/g) at 1 h postinjection than in the PSMA negative PC3-Flu tumors (1.12 ± 0.42%ID/g). Taken together, we have successfully designed and synthesized a T-SMDC system for prostate cancer targeted imaging and therapy.
Assuntos
Desenho de Fármacos , Imunoconjugados/metabolismo , Imunoconjugados/uso terapêutico , Terapia de Alvo Molecular , Tomografia por Emissão de Pósitrons combinada à Tomografia Computadorizada/métodos , Neoplasias da Próstata/diagnóstico por imagem , Neoplasias da Próstata/terapia , Antígenos de Superfície/metabolismo , Linhagem Celular Tumoral , Estabilidade de Medicamentos , Radioisótopos de Gálio , Glutamato Carboxipeptidase II/metabolismo , Humanos , Imunoconjugados/química , Masculino , Polietilenoglicóis/química , Neoplasias da Próstata/patologiaRESUMO
Bavituximab is a chimeric monoclonal antibody with immune modulating and tumor-associated vascular disrupting properties demonstrated in models of non-small cell lung cancer (NSCLC). The molecular target of Bavituximab, phosphatidylserine (PS), is exposed on the outer leaflet of the membrane bi-layer of malignant vascular endothelial cells and tumor cells to a greater extent than on normal tissues. We evaluated the tumor-targeting properties of Bavituximab for imaging of NSCLC xenografts when radiolabeled with (111)In through conjugation with a bifunctional chelating agent, 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA). In vitro binding of (111)In-DOTA-Bavituximab to PS was determined by enzyme-linked immunosorbent assay (ELISA). Biodistribution of (111)In-DOTA-Bavituximab was conducted in normal rats, which provided data for dosimetry calculation. Single-photon emission computed tomography/computed tomography (SPECT/CT) imaging was performed in athymic nude rats bearing A549 NSCLC xenografts. At the molar conjugation ratio of 0.54 DOTA per Bavituximab, the PS binding affinity of (111)In-DOTA-Bavituximab was comparable to that of unmodified Bavituximab. Based on the quantitative SPECT/CT imaging data analysis, (111)In-DOTA-Bavituximab demonstrated tumor-specific uptake as measured by the tumor-tomuscle ratio, which peaked at 5.2 at 72 hr post-injection. In contrast, the control antibody only presented a contrast of 1.2 at the same time point.These findings may underlie the diagnostic efficacy and relative low rates of systemic vascular and immune-related toxicities of this immunoconjugate. Future applications of (111)In-DOTA-bavituximab may include prediction of efficacy, indication of tumor immunologic status, or characterization of radiographic findings.
RESUMO
The goal of this study is to evaluate a new (68) Ga-based imaging agent for detecting tumor hypoxia using positron emission tomography (PET). The new hypoxia targeting agent reported here, [(68) Ga]-HP-DO3A-nitroimidazole ([(68) Ga]-HP-DO3A-NI), was constructed by linking a nitroimidazole moiety with the macrocyclic ligand component of ProHance®, HP-DO3A. The hypoxia targeting capability of this agent was evaluated in A549 lung cancer cells in vitro and in SCID mice bearing subcutaneous A549 tumor xenografts. The cellular uptake assays showed that significantly more [(68) Ga]-HP-DO3A-NI accumulates in hypoxic tumor cells at 30, 60 and 120 min than in the same cells exposed to 21% O2 . The agent also accumulated in hypoxic tumors in vivo to give a tumor/muscle ratio (T/M) of 5.0 ± 1.2 (n = 3) as measured by PET at 2 h post-injection (p.i.). This was further confirmed by ex vivo biodistribution data. In addition, [(68) Ga]-HP-DO3A-NI displayed very favorable pharmacokinetic properties, as it was cleared largely through the kidneys with little to no accumulation in liver, heart or lung (%ID/g < 0.5%) at 2 h p.i. The specificity of the agent for hypoxic tissues was further validated in a comparative study with a control compound, [(68) Ga]-HP-DO3A, which lacks the nitroimidazole moiety, and by PET imaging of tumor-bearing mice breathing air versus 100% O2 . Given the commercial availability of cGMP (68) Ge/(68) Ga generators and the ease of (68) Ga labeling, the new agent could potentially be widely applied for imaging tumor hypoxia prior to radiation therapy.
Assuntos
Hipóxia Celular/fisiologia , Meios de Contraste/química , Radioisótopos de Gálio/química , Compostos Heterocíclicos com 1 Anel/química , Neoplasias Pulmonares/diagnóstico por imagem , Nitroimidazóis/química , Tomografia por Emissão de Pósitrons/métodos , Animais , Transporte Biológico/fisiologia , Linhagem Celular Tumoral , Feminino , Humanos , Marcação por Isótopo/métodos , Camundongos , Camundongos SCID , Transplante de Neoplasias , Compostos Radiofarmacêuticos/farmacocinética , Transplante HeterólogoRESUMO
We report a click-chemistry based modular strategy for antibody labeling with (64)Cu (t1/2 = 12.7 h; ß(+) 0.656 MeV, 17.4%; ß(-) 0.573 MeV, 39%; EC 43%) under ambient condition utilizing a cross-bridged tetraazamacrocyclic (CB-TE2A) analogue, which otherwise requires harsh conditions that make the CB-TE2A analogues under-utilized for protein labeling despite the fact that they form kinetically inert copper complexes with high in vivo stability. Our strategy involves prelabeling a CB-TE2A based scaffold (CB-TE2A-1C) with (64)Cu and its subsequent reaction with an antibody via the tetrazine-norbornene mediated click chemistry. The effectiveness of this strategy was demonstrated by labeling two monoclonal antibodies, an anti-PSMA antibody (YPSMA-1) and a chimeric anti-phosphatidylserine antibody (Bavituximab). The immunoreactivity of the antibodies remained unchanged after the tetrazine modification and click-chemistry (64)Cu labeling. To further demonstrate the practicality of the modular (64)Cu labeling strategy, we tested positron emission tomography (PET) imaging of tumor with the (64)Cu-labeled bavituximab in a mouse xenograft model. The tumor visualization and uptake of the labeled antibody exhibited the versatility of the click-chemistry strategy.
Assuntos
Adenocarcinoma/diagnóstico por imagem , Anticorpos Monoclonais/química , Quelantes/síntese química , Radioisótopos de Cobre/química , Compostos Heterocíclicos com 1 Anel/síntese química , Neoplasias da Próstata/diagnóstico por imagem , Compostos Radiofarmacêuticos/química , Adenocarcinoma/genética , Adenocarcinoma/ultraestrutura , Animais , Anticorpos Monoclonais/farmacocinética , Antígenos de Superfície/genética , Antígenos de Superfície/metabolismo , Quelantes/química , Química Click , Expressão Gênica , Glutamato Carboxipeptidase II/antagonistas & inibidores , Glutamato Carboxipeptidase II/genética , Glutamato Carboxipeptidase II/metabolismo , Compostos Heterocíclicos com 1 Anel/química , Humanos , Masculino , Camundongos , Tomografia por Emissão de Pósitrons , Neoplasias da Próstata/genética , Neoplasias da Próstata/ultraestrutura , Coloração e Rotulagem/métodos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Degradation of inorganic nanoparticles (NPs) into small molecular complexes is often observed in the physiological environment; however, how this process influences renal clearance of inorganic NPs is largely unknown. By systematically comparing renal clearance of degradable luminescent glutathione coated copper NPs (GS-CuNPs) and their dissociated products, Cu(II)-glutathione disulfide (GSSG) complexes (Cu(II)-GSSG), we found that GS-CuNPs were eliminated through the urinary system surprisingly faster and accumulated in the liver much less than their smaller dissociation counterparts. With assistance of radiochemistry and positron emission tomography (PET) imaging, we found that the observed "nano size" effect in enhancing renal clearance is attributed to the fact that GS-CuNPs are more resistant to serum protein adsorption than Cu(II)-GSSG. In addition, since dissociation of GS-CuNPs follows zero-order chemical kinetics, their renal clearance and biodistribution also depend on initial injection doses and their dissociation processes. Quantitative understanding of size effect and other factors involved in renal clearance and biodistribution of degradable inorganic NPs will lay down a foundation for further development of renal-clearable inorganic NPs with minimized nanotoxicity.
Assuntos
Cobre/metabolismo , Glutationa/metabolismo , Rim/metabolismo , Taxa de Depuração Metabólica/fisiologia , Nanopartículas Metálicas , Animais , Cobre/farmacologia , Glutationa/farmacologia , Rim/efeitos dos fármacos , Taxa de Depuração Metabólica/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos BALB CRESUMO
We report a versatile dendritic structure based platform for construction of targeted dual-modality imaging probes. The platform contains multiple copies of 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) branching out from a 1,4,7-triazacyclononane-N,N',Nâ³-triacetic acid (NOTA) core. The specific coordination chemistries of the NOTA and DOTA moieties offer specific loading of (68/67)Ga(3+) and Gd(3+), respectively, into a common molecular scaffold. The platform also contains three amino groups which can potentiate targeted dual-modality imaging of PET/MRI or SPECT/MRI (PET: positron emission tomography; SPECT: single photon emission computed tomography; MRI: magnetic resonance imaging) when further functionalized by targeting vectors of interest. To validate this design concept, a bimetallic complex was synthesized with six peripheral Gd-DOTA units and one Ga-NOTA core at the center, whose ion T1 relaxivity per gadolinium atom was measured to be 15.99 mM(-1) s(-1) at 20 MHz. Further, the bimetallic agent demonstrated its anticipated in vivo stability, tissue distribution, and pharmacokinetic profile when labeled with (67)Ga. When conjugated with a model targeting peptide sequence, the trivalent construct was able to visualize tumors in a mouse xenograft model by both PET and MRI via a single dose injection.
Assuntos
Desenho de Fármacos , Corantes Fluorescentes/síntese química , Gadolínio/química , Radioisótopos de Gálio/química , Animais , Linhagem Celular Tumoral , Corantes Fluorescentes/análise , Corantes Fluorescentes/metabolismo , Gadolínio/análise , Gadolínio/metabolismo , Radioisótopos de Gálio/análise , Radioisótopos de Gálio/metabolismo , Humanos , Isótopos , Imageamento por Ressonância Magnética/métodos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos SCID , Tomografia por Emissão de Pósitrons/métodos , Ratos , Distribuição Tecidual/fisiologia , Ensaios Antitumorais Modelo de Xenoenxerto/métodosRESUMO
The macrocyclic bone-seeking agent, DO2A2P, bears a cyclen core and two pairs of peripheral phosphonate and carboxylate groups. The geometric disposition of the peripheral functionalities gives arise to a pair of geometric isomers: cis-DO2A2P and trans-DO2A2P. In order to compare the biological behavior of the isomer pair, cis-DO2A2P was synthesized. Both isomers were successfully radiolabeled with (177)Lu, which might potentiate their applications in both radiotherapy and imaging of bone diseases. Through a set of biological assays including the hydroxyapatite binding, in vitro stability, and in vivo distribution, we demonstrated that the geometric pair of DO2A2P had virtually identical biological properties.