RESUMO
Glioblastoma (GBM) is the most common and malignant primary brain tumor in adults. Immunotherapy may be promising for the treatment of some patients with GBM; however, there is a need for noninvasive neuroimaging techniques to predict immunotherapeutic responses. The effectiveness of most immunotherapeutic strategies requires T-cell activation. Therefore, we aimed to evaluate an early marker of T-cell activation, CD69, for its use as an imaging biomarker of response to immunotherapy for GBM. Herein, we performed CD69 immunostaining on human and mouse T cells following in vitro activation and post immune checkpoint inhibitors (ICI) in an orthotopic syngeneic mouse glioma model. CD69 expression on tumor-infiltrating leukocytes was assessed using single-cell RNA sequencing (scRNA-seq) data from patients with recurrent GBM receiving ICI. Radiolabeled CD69 Ab PET/CT imaging (CD69 immuno-PET) was performed on GBM-bearing mice longitudinally to quantify CD69 and its association with survival following immunotherapy. We show CD69 expression is upregulated upon T-cell activation and on tumor-infiltrating lymphocytes (TIL) in response to immunotherapy. Similarly, scRNA-seq data demonstrated elevated CD69 on TILs from patients with ICI-treated recurrent GBM as compared with TILs from control cohorts. CD69 immuno-PET studies showed a significantly higher tracer uptake in the tumors of ICI-treated mice compared with controls. Importantly, we observed a positive correlation between survival and CD69 immuno-PET signals in immunotherapy-treated animals and established a trajectory of T-cell activation by virtue of CD69-immuno-PET measurements. Our study supports the potential use of CD69 immuno-PET as an immunotherapy response assessment imaging tool for patients with GBM. Significance: Immunotherapy may hold promise for the treatment of some patients with GBM. There is a need to assess therapy responsiveness to allow the continuation of effective treatment in responders and to avoid ineffective treatment with potential adverse effects in the nonresponders. We demonstrate that noninvasive PET/CT imaging of CD69 may allow early detection of immunotherapy responsiveness in patients with GBM.
Assuntos
Glioblastoma , Animais , Humanos , Camundongos , Glioblastoma/diagnóstico por imagem , Imunoterapia , Recidiva Local de Neoplasia , Tomografia por Emissão de Pósitrons combinada à Tomografia Computadorizada , Tomografia por Emissão de Pósitrons/métodos , Linfócitos T/metabolismoRESUMO
Significance: This third biennial intraoperative molecular imaging (IMI) conference shows how optical contrast agents have been applied to develop clinically significant endpoints that improve precision cancer surgery. Aim: National and international experts on IMI presented ongoing clinical trials in cancer surgery and preclinical work. Previously known dyes (with broader applications), new dyes, novel nonfluorescence-based imaging techniques, pediatric dyes, and normal tissue dyes were discussed. Approach: Principal investigators presenting at the Perelman School of Medicine Abramson Cancer Center's third clinical trials update on IMI were selected to discuss their clinical trials and endpoints. Results: Dyes that are FDA-approved or currently under clinical investigation in phase 1, 2, and 3 trials were discussed. Sections on how to move benchwork research to the bedside were also included. There was also a dedicated section for pediatric dyes and nonfluorescence-based dyes that have been newly developed. Conclusions: IMI is a valuable adjunct in precision cancer surgery and has broad applications in multiple subspecialties. It has been reliably used to alter the surgical course of patients and in clinical decision making. There remain gaps in the utilization of IMI in certain subspecialties and potential for developing newer and improved dyes and imaging techniques.
Assuntos
Neoplasias , Humanos , Criança , Neoplasias/diagnóstico por imagem , Neoplasias/cirurgia , Meios de Contraste , Imagem Molecular/métodos , CorantesRESUMO
18F-fluorodeoxyglucose (FDG) is a glucose analog that acts as a marker for glucose uptake and metabolism. FDG PET scans are used in monitoring pediatric cancers. The handheld PET probe localization of FDG-avid lesions is an emerging modality for radio-guided surgery (RGS). We sought to assess the utility of PET probe in localizing occult FDG-avid tumors in pediatric patients. PET probe functionality was evaluated by using a PET/CT scan calibration phantom. The PET probe was able to detect FDG photon emission from simulated tumors with an expected decay of the radioisotope over time. Specificity for simulated tumor detection was lower in a model that included background FDG. In a clinical model, eight pediatric patients with FDG-avid primary, recurrent or metastatic cancer underwent a tumor excision, utilizing IV FDG and PET probe survey. Adequate tissue for diagnosis was present in 16 of 17 resected specimens, and pathology was positive for malignancy in 12 of the 17 FDG-avid lesions. PET probe gamma counts per second were higher in tumors compared with adjacent benign tissue in all operations. The median ex vivo tumor-to-background ratio (TBR) was 4.0 (range 0.9-12). The PET probe confirmed the excision of occult FDG-avid tumors in eight pediatric patients.
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Pramlintide is an equipotent amylin analogue that reduces food intake and body weight in obese subjects and has been clinically approved as an adjunctive therapy for the treatment of adult diabetic patients. However, due to its extremely short half-life in vivo, a regimen of multiple daily administrations is required for achieving clinical effectiveness. Herein is described the development of prototypical long-acting pramlintide bioconjugates, in which pramlintide's disulfide-linked macrocycle was replaced by a cyclic thioether motif. This modification enabled stable chemical conjugation to a half-life extending antibody. In contrast to pramlintide (t1/2 < 0.75 h), bioconjugates 35 and 38 have terminal half-lives of â¼2 days in mice and attain significant exposure levels that are maintained up to 7 days. Single dose subcutaneous administration of 35 in lean mice, given 18-20 h prior to oral acetaminophen (AAP) administration, significantly reduced gastric emptying (as determined by plasma AAP levels). In a separate study, similar administration of 35 in fasted lean mice effected a reduction in food intake for up to 48 h. These data are consistent with durable amylinomimetic responses and provide the basis for further development of such long-acting amylinomimetic conjugates for the potential treatment of obesity and associated pathologies.
Assuntos
Agonistas dos Receptores da Amilina , Agonistas dos Receptores da Amilina/farmacologia , Agonistas dos Receptores da Amilina/uso terapêutico , Amiloide , Animais , Peso Corporal , Humanos , Hipoglicemiantes/uso terapêutico , Polipeptídeo Amiloide das Ilhotas Pancreáticas/farmacologia , Camundongos , Obesidade/induzido quimicamente , Obesidade/tratamento farmacológicoRESUMO
The long circulating half-life and inherently bivalent architecture of IgGs provide an ideal vehicle for presenting otherwise short-lived G-protein-coupled receptor agonists in a format that enables avidity-driven enhancement of potency. Here, we describe the site-specific conjugation of a dual agonist peptide (an oxyntomodulin variant engineered for potency and in vivo stability) to the complementarity-determining regions (CDRs) of an immunologically silent IgG4. A cysteine-containing heavy chain CDR3 variant was identified that provided clean conjugation to a bromoacetylated peptide without interference from any of the endogenous mAb cysteine residues. The resulting mAb-peptide homodimer has high potency at both target receptors (glucagon receptor, GCGR, and glucagon-like peptide 1 receptor, GLP-1R) driven by an increase in receptor avidity provided by the spatially defined presentation of the peptides. Interestingly, the avidity effects are different at the two target receptors. A single dose of the long-acting peptide conjugate robustly inhibited food intake and decreased body weight in insulin resistant diet-induced obese mice, in addition to ameliorating glucose intolerance. Inhibition of food intake and decrease in body weight was also seen in overweight cynomolgus monkeys. The weight loss resulting from dosing with the bivalently conjugated dual agonist was significantly greater than for the monomeric analog, clearly demonstrating translation of the measured in vitro avidity to in vivo pharmacology.
Assuntos
Anticorpos Monoclonais , Ingestão de Alimentos/efeitos dos fármacos , Obesidade , Oxintomodulina , Peptídeos , Animais , Anticorpos Monoclonais/química , Anticorpos Monoclonais/farmacocinética , Anticorpos Monoclonais/farmacologia , Cisteína/química , Células HEK293 , Humanos , Macaca fascicularis , Masculino , Camundongos , Obesidade/sangue , Obesidade/tratamento farmacológico , Oxintomodulina/química , Oxintomodulina/farmacocinética , Oxintomodulina/farmacologia , Peptídeos/química , Peptídeos/farmacocinética , Peptídeos/farmacologiaRESUMO
The gut hormone PYY3-36 reduces food intake in humans and exhibits at least additive efficacy in combination with GLP-1. However, the utility of PYY analogs as anti-obesity agents has been severely limited by emesis and rapid proteolysis, a profile similarly observed with native PYY3-36 in obese rhesus macaques. Here, we found that antibody conjugation of a cyclized PYY3-36 analog achieved high NPY2R selectivity, unprecedented in vivo stability, and gradual infusion-like exposure. These properties permitted profound reduction of food intake when administered to macaques for 23 days without a single emetic event in any animal. Co-administration with the GLP-1 receptor agonist liraglutide for an additional 5 days further reduced food intake with only one animal experiencing a single bout of emesis. This antibody-conjugated PYY analog therefore may enable the long-sought potential of GLP-1/PYY-based combination treatment to achieve robust, well-tolerated weight reduction in obese patients.
Assuntos
Anorexia/induzido quimicamente , Peptídeo YY/química , Peptídeo YY/farmacologia , Vômito , Animais , Células CHO , Cricetulus , Receptor do Peptídeo Semelhante ao Glucagon 1/agonistas , Receptor do Peptídeo Semelhante ao Glucagon 1/metabolismo , Células HEK293 , Humanos , Liraglutida/farmacologia , Macaca mulatta , Camundongos , Camundongos Endogâmicos C57BL , Obesidade/tratamento farmacológico , Obesidade/metabolismo , Peptídeo YY/administração & dosagem , Vômito/induzido quimicamenteRESUMO
Murine antibody 10H10 raised against human tissue factor is unique in that it blocks the signaling pathway, and thus inhibits angiogenesis and tumor growth without interfering with coagulation. As a potential therapeutic, the antibody was humanized in a two-step procedure. Antigen-binding loops were grafted onto selected human frameworks and the resulting chimeric antibody was subjected to affinity maturation by using phage display libraries. The results of humanization were analyzed from the structural perspective through comparison of the structure of a humanized variant with the parental mouse antibody. This analysis revealed several hot spots in the framework region that appear to affect antigen binding, and therefore should be considered in human germline selection. In addition, some positions in the Vernier zone, e.g., residue 71 in the heavy chain, that are traditionally thought to be crucial appear to tolerate amino acid substitutions without any effect on binding. Several humanized variants were produced using both short and long forms of complementarity-determining region (CDR) H2 following the difference in the Kabat and Martin definitions. Comparison of such pairs indicated consistently higher thermostability of the variants with short CDR H2. Analysis of the binding data in relation to the structures singled out the ImMunoGeneTics information system® germline IGHV1-2*01 as dubious owing to two potentially destabilizing mutations as compared to the other alleles of the same germline and to other human germlines.
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Anticorpos Monoclonais Humanizados/química , Afinidade de Anticorpos/fisiologia , Tromboplastina/imunologia , Animais , Anticorpos Monoclonais Humanizados/imunologia , Regiões Determinantes de Complementaridade/química , Humanos , Camundongos , Modelos Moleculares , Engenharia de Proteínas/métodosRESUMO
INTRODUCTION: Imaging fatty acid uptake and utilization has broad impact in investigating myocardial diseases, hepatic functions, tumor progression, and the metabolic state of adipose tissue. The SPECT tracer (123)I-15-(p-iodophenyl)-3-(R,S)-methylpentadecanoic acid (BMIPP) is a clinically used nuclear medicine tracer to image myocardial uptake of fatty acid. Although ((18)F-5) has been in clinical use for PET imaging of adipose tissue as well as the myocardium, here we developed a click oleate analog to compare to FTO, with the goal of improved stability to defluorination and suitability for imaging myocardial uptake and oxidation of fatty acids. METHODS: A rapid and convenient synthetic approach for a precursor to a (18)F-labeled oleate analog using click chemistry was developed and evaluated for PET imaging in fasted mice. RESULTS: The overall yield for the preparation of the labeling precursor of the clicked oleate analog was 12%. This precursor was efficiently radiolabeled with F-18 in 17% non-decay-corrected radiochemical yield. PET/CT imaging and biodistribution results show that this fatty acid analog had reasonable heart uptake (0.94±0.28 %ID/g at 0.5 h p.i.) and heart-to-muscle ratio (2.05±0.39 at 0.5h p.i.) and is a potential lead for developing new PET tracers to image fatty acid uptake and utilization using click chemistry methodologies. The synthetic route to FTO was optimized to three steps from known starting materials. CONCLUSION: While the uptake of the clicked oleic acid analog was sufficient for visualizing the myocardium in mice, the preliminary metabolism data suggest that only a fraction of the uptake was due to fatty acid beta-oxidation. Studies are under way to explore the uptake/oxidation mechanism and kinetics.
Assuntos
Ácidos Graxos/metabolismo , Radioisótopos de Flúor , Ácido Oleico/síntese química , Tomografia por Emissão de Pósitrons/métodos , Animais , Transporte Biológico , Técnicas de Química Sintética , Marcação por Isótopo , Masculino , Camundongos , Ácido Oleico/química , Ácido Oleico/farmacocinética , Distribuição Tecidual , Tomografia Computadorizada por Raios XRESUMO
Matrix metalloproteinases (MMP) 2 and 9, the gelatinases, have consistently been associated with tumor progression. The development of gelatinase-specific probes will be critical for identifying in vivo gelatinoic activity to understand the molecular role of the gelatinases in tumor development. Recently, a self-assembling homotrimeric triple-helical peptide (THP), incorporating a sequence from type V collagen, with high substrate specificity to the gelatinases has been developed. To determine whether this THP would be suitable for imaging protease activity, 5-carboxyfluorescein (5FAM) was conjugated, resulting in 5FAM3-THP and 5FAM6-THP, which were quenched up to 50%. 5FAM6-THP hydrolysis by MMP-2 and MMP-9 displayed kcat/KM values of 1.5 × 104 and 5.4 × 103 M-1 s-1, respectively. Additionally 5FAM6-THP visualized gelatinase activity in gelatinase positive HT-1080 cells, but not in gelatinase negative MCF-7 cells. Furthermore, the fluorescence in the HT-1080 cells was greatly attenuated by the addition of a MMP-2 and MMP-9 inhibitor, SB-3CT, indicating that the observed fluorescence release was mediated by gelatinase proteolysis and not non-specific proteolysis of the THPs. These results demonstrate that THPs fully substituted with fluorophores maintain their substrate specificity to the gelatinases in human cancer cells and may be useful in in vivo molecular imaging of gelatinase activity.
Assuntos
Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Peptídeos/farmacocinética , Tomografia Óptica/métodos , Linhagem Celular Tumoral , Colágeno Tipo V/química , Fluoresceínas/química , Fluorescência , Corantes Fluorescentes/química , Humanos , Células MCF-7 , Microscopia Confocal , Microscopia de Fluorescência , Peptídeos/síntese química , Peptídeos/químicaRESUMO
Ion channels are an attractive class of drug targets, but progress in developing inhibitors for therapeutic use has been limited largely due to challenges in identifying subtype selective small molecules. Animal venoms provide an alternative source of ion channel modulators, and the venoms of several species, such as scorpions, spiders and snails, are known to be rich sources of ion channel modulating peptides. Importantly, these peptides often bind to hyper-variable extracellular loops, creating the potential for subtype selectivity rarely achieved with small molecules. We have engineered scorpion venom peptides and incorporated them in fusion proteins to generate highly potent and selective Kv1.3 inhibitors with long in vivo half-lives. Kv1.3 has been reported to play a role in human T cell activation, and therefore, these Kv1.3 inhibitor fusion proteins may have potential for the treatment of autoimmune diseases. Our results support an emerging approach to generating subtype selective therapeutic ion channel inhibitors.