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INTRODUCTION: Amyloid deposition is a cause of restrictive cardiomyopathy. Patients who present with cardiac disease can be evaluated for transthyretin (TTR)-associated cardiac amyloidosis using nuclear imaging with 99mTc-labeled pyrophosphate (PYP); however, light chain-associated (AL) cardiac amyloid is generally not detected using this tracer. As an alternative, the amyloid-binding peptide p5+14 radiolabeled with iodine-124 has been shown to be an effective pan-amyloid radiotracer for PET/CT imaging. Here, a 99mTc-labeled form of p5+14 peptide has been prepared to facilitate SPECT/CT imaging of cardiac amyloidosis. METHOD: A synthesis method suitable for clinical applications has been used to prepare 99mTc-labeled p5+14 and tested for peptide purity, product bioactivity, radiochemical purity and stability. The product was compared with99mTc-PYP for cardiac SPECT/CT imaging in a mouse model of AA amyloidosis and for reactivity with human tissue sections from AL and TTR patients. RESULTS: The 99mTc p5+14 tracer was produced with >95% yields in radiopurity and bioactivity with no purification steps required and retained over 95% peptide purity and >90% bioactivity for >3 h. In mice, the tracer detected hepatosplenic AA amyloid as well as heart deposits with uptake ~5 fold higher than 99mTc-PYP. 99mTc p5+14 effectively bound human amyloid deposits in the liver, kidney and both AL- and ATTR cardiac amyloid in tissue sections in which 99mTc-PYP binding was not detectable. CONCLUSION: 99mTc-p5+14 was prepared in minutes in >20 mCi doses with good performance in preclinical studies making it suitable for clinical SPECT/CT imaging of cardiac amyloidosis.
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Amiloidosis , Cardiomiopatías , Humanos , Ratones , Animales , Tomografía Computarizada por Tomografía de Emisión de Positrones , Amiloidosis/diagnóstico por imagen , Amiloidosis/metabolismo , Tomografía Computarizada de Emisión de Fotón Único/métodos , Péptidos , Amiloide/metabolismo , Cardiomiopatías/diagnóstico por imagen , PrealbúminaRESUMEN
BACKGROUND: The noninvasive detection of cardiac amyloid, as well as deposits in other vital organs, is critical for early diagnosis and quantitative disease monitoring. Positron emission tomography is an intrinsically quantitative imaging modality suitable for high-resolution amyloid detection. OBJECTIVES: This study sought to evaluate the safety and efficacy of a novel amyloid-reactive peptide, designated p5+14, labeled with iodine-124 (124I), in patients with diverse types of systemic amyloidosis. METHODS: In a single-site, open label phase 1/2 study (NCT03678259), the safety, biodistribution, and sensitivity of a single intravenous infusion of 124I-evuzamitide was assessed in patients with systemic amyloidosis (n = 50), asymptomatic transthyretin sequence variant carriers (n = 2), and healthy volunteers (n = 5). Subjects were administered 1.4 ± 0.2 mg of 124I-evuzamitide (71.5 ± 12.4 MBq) and positron emission tomography/x-ray computed tomography images acquired at 5.2 hours (Q25-Q75: 4.9-5.4 hours) postinfusion. Images were assessed visually and semi-quantitatively for positive uptake of radiotracer in the heart and other major organs. RESULTS: Uptake of 124I-evuzamitide in the heart and other abdominothoracic organs was consistent with the patient's clinical presentation and the type of amyloidosis. The patient- and cardiac-associated sensitivity for imaging and clinical observations was 93.6% (95% CI: 82.8%-97.8%) and 96.2% (95% CI: 81.8%-99.8%), respectively. Semi-quantitative uptake of the radiotracer correlated significantly with serum N-terminal pro-B-type natriuretic peptide measurements in patients with light chain-associated amyloidosis. Cardiac uptake was not observed in any healthy volunteers. The agent was well tolerated, with 1 drug-related adverse event and no deaths. CONCLUSIONS: 124I-evuzamitide is an amyloid-binding radiotracer capable of detecting cardiac amyloid in patients with high sensitivity.
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Amiloidosis , Amiloidosis de Cadenas Ligeras de las Inmunoglobulinas , Humanos , Tomografía Computarizada por Tomografía de Emisión de Positrones , Distribución Tisular , Valor Predictivo de las Pruebas , Amiloide , Radioisótopos de Yodo , Amiloidosis/diagnóstico por imagenRESUMEN
Introduction: Systemic amyloidosis is a progressive disorder characterized by the extracellular deposition of amyloid fibrils and accessory proteins in visceral organs and tissues. Amyloid accumulation causes organ dysfunction and is not generally cleared by the immune system. Current treatment focuses on reducing amyloid precursor protein synthesis and slowing amyloid deposition. However, curative interventions will likely also require removal of preexisting amyloid deposits to restore organ function. Here we describe a prototypic pan-amyloid binding peptide-antibody fusion molecule (mIgp5) that enhances macrophage uptake of amyloid. Methods: The murine IgG1-IgG2a hybrid immunoglobulin with a pan amyloid-reactive peptide, p5, fused genetically to the N-terminal of the immunoglobulin light chain was synthesized in HEK293T/17 cells. The binding of the p5 peptide moiety was assayed using synthetic amyloid-like fibrils, human amyloid extracts and amyloid-laden tissues as substrates. Binding of radioiodinated mIgp5 with amyloid deposits in vivo was evaluated in a murine model of AA amyloidosis using small animal imaging and microautoradiography. The bioactivity of mIgp5 was assessed in complement fixation and in vitro phagocytosis assays in the presence of patient-derived amyloid extracts and synthetic amyloid fibrils as substrates and in the presence or absence of human serum. Results: Murine Igp5 exhibited highly potent binding to AL and ATTR amyloid extracts and diverse types of amyloid in formalin-fixed tissue sections. In the murine model of systemic AA amyloidosis, 125I-mIgp5 bound rapidly and specifically to amyloid deposits in all organs, including the heart, with no evidence of non-specific uptake in healthy tissues. The bioactivity of the immunoglobulin Fc domain was uncompromised in the context of mIgp5 and served as an effective opsonin. Macrophage-mediated uptake of amyloid extract and purified amyloid fibrils was enhanced by the addition of mIgp5. This effect was exaggerated in the presence of human serum coincident with deposition of complement C5b9. Conclusion: Immunostimulatory, amyloid-clearing therapeutics can be developed by incorporating pan-amyloid-reactive peptides, such as p5, as a targeting moiety. The immunologic functionality of the IgG remains intact in the context of the fusion protein. These data highlight the potential use of peptide-antibody fusions as therapeutics for all types of systemic amyloidosis.
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Amiloidosis , Placa Amiloide , Ratones , Animales , Humanos , Modelos Animales de Enfermedad , Células HEK293 , Amiloidosis/metabolismo , Amiloide/metabolismo , Proteínas Amiloidogénicas/metabolismo , Péptidos/metabolismo , Cadenas Ligeras de InmunoglobulinaRESUMEN
There are at least 20 distinct types of systemic amyloidosis, all of which result in the organ-compromising accumulation of extracellular amyloid deposits. Amyloidosis is challenging to diagnose due to the heterogeneity of the clinical presentation, yet early detection is critical for favorable patient outcomes. The ability to non-invasively and quantitatively detect amyloid throughout the body, even in at-risk populations, before clinical manifestation would be invaluable. To this end, a pan-amyloid-reactive peptide, p5+14, has been developed that is capable of binding all types of amyloid. Herein, we demonstrate the ex vivo pan-amyloid reactivity of p5+14 by using peptide histochemistry on animal and human tissue sections containing various types of amyloid. Furthermore, we present clinical evidence of pan-amyloid binding using iodine-124-labeled p5+14 in a cohort of patients with eight (n = 8) different types of systemic amyloidosis. These patients underwent PET/CT imaging as part of the first-in-human Phase 1/2 clinical trial evaluating this radiotracer (NCT03678259). The uptake of 124I-p5+14 was observed in abdominothoracic organs in patients with all types of amyloidosis evaluated and was consistent with the disease distribution described in the medical record and literature reports. On the other hand, the distribution in healthy subjects was consistent with radiotracer catabolism and clearance. The early and accurate diagnosis of amyloidosis remains challenging. These data support the utility of 124I-p5+14 for the diagnosis of varied types of systemic amyloidosis by PET/CT imaging.
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BACKGROUND: Systemic amyloidosis refers to a group of protein misfolding disorders characterized by the extracellular deposition of amyloid fibrils in organs and tissues. For reasons heretofore unknown, amyloid deposits are not recognized by the immune system, and progressive deposition leads to organ dysfunction. METHODS: In vitro and in vivo phagocytosis assays were performed to elucidate the impact of collagen and other amyloid associated proteins (eg serum amyloid p component and apolipoprotein E) had on amyloid phagocytosis. Immunohistochemical and histopathological staining regimens were employed to analyze collagen-amyloid interactions and immune responses. RESULTS: Histological analysis of amyloid-laden tissue indicated that collagen is intimately associated with amyloid deposits. We report that collagen inhibits phagocytosis of amyloid fibrils by macrophages. Treatment of 15 patient-derived amyloid extracts with collagenase significantly enhanced amyloid phagocytosis. Preclinical mouse studies indicated that collagenase treatment of amyloid extracts significantly enhanced clearance as compared to controls, coincident with increased immune cell infiltration of the subcutaneous amyloid lesion. CONCLUSIONS: These data suggest that amyloid-associated collagen serves as a 'don't eat me' signal, thereby hindering clearance of amyloid. Targeted degradation of amyloid-associated collagen could result in innate immune cell recognition and clearance of pathologic amyloid deposits.
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Amiloide , Placa Amiloide , Animales , Ratones , Amiloide/metabolismo , Placa Amiloide/metabolismo , Fagocitosis/fisiología , Macrófagos/metabolismo , Proteínas Amiloidogénicas/metabolismo , Colágeno/metabolismoRESUMEN
PURPOSE: Accurate diagnosis of amyloidosis remains a significant clinical challenge and unmet need for patients. The amyloid-reactive peptide p5+14 radiolabeled with iodine-124 has been developed for the detection of amyloid by PET/CT imaging. In a first-in-human evaluation, the dosimetry and tissue distribution of 124I-p5+14 peptide in patients with systemic amyloidosis. Herein, we report the dosimetry and dynamic distribution in the first three enrolled patients with light chain-associated (AL) amyloidosis. PROCEDURES: The radiotracer was assessed in a single-site, open-label phase 1 study (NCT03678259). The first three patients received a single intravenous infusion of 124I-p5+14 peptide (≤37 MBq). Serial PET/CT imaging was performed during the 48 h post-infusion. Dosimetry was determined as a primary endpoint for each patient and gender-averaged mean values were calculated. Pharmacokinetic parameters were estimated from whole blood radioactivity measurements and organ-based time activity data. Lastly, the biodistribution of radiotracer in major organs was assessed visually and compared to clinically appreciated organ involvement. RESULTS: Infusion of the 124I-p5+14 was well tolerated with rapid uptake in the heart, kidneys, liver, spleen, pancreas, and lung. The gender-averaged whole-body effective radiation dose was estimated to be 0.23 (± 0.02) mSv/MBq with elimination of the radioactivity via renal and gastrointestinal routes. The whole blood elimination t1/2 of 21.9 ± 7.6 h. Organ-based activity concentration measurements indicated that AUClast tissue:blood ratios generally correlated with the anticipated presence of amyloid. Peptide uptake was observed in 4/5 clinically suspected organs, as noted in the medical record, as well as six anatomic sites generally associated with amyloidosis in this population. CONCLUSION: Peptide 124I-p5+14 rapidly distributes to anatomic sites consistent with the presence of amyloid in patients with systemic AL. The dosimetry estimates established in this cohort are acceptable for whole-body PET/CT imaging. Pharmacokinetic parameters are heterogeneous and consistent with uptake of the tracer in an amyloid compartment. PET/CT imaging of 124I-p5+14 may facilitate non-invasive detection of amyloid in multiple organ systems.
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Amiloidosis , Amiloidosis de Cadenas Ligeras de las Inmunoglobulinas , Amiloide/metabolismo , Amiloidosis/diagnóstico por imagen , Humanos , Radioisótopos de Yodo , Péptidos , Tomografía Computarizada por Tomografía de Emisión de Positrones , Tomografía de Emisión de Positrones/métodos , Radiometría , Distribución TisularRESUMEN
Systemic light chain amyloidosis (AL) causes a malignant pathology associated with the formation of amyloid fibrils that deposit in human organs and tissues, leading to dysfunction and severe morbidity. Amyloid fibril-reactive antibodies have been used to remove amyloid from organs and are effective in restoring organ function in patients with AL amyloidosis. Unfortunately, antibodies do not bind amyloid in all AL patients, nor do they efficiently bind many other forms of amyloid. Recently, a synthetic peptide P62 was developed, which binds many forms of systemic amyloidosis and can be further modified and fused to a high-affinity peptide epitope to expand its utility as a novel amyloid immunotherapeutic. However, the molecular-level details of P62-fibril binding mechanisms, critical for future peptide design, are unclear. Here, we combine protein docking, all-atom molecular dynamics simulation and umbrella sampling to study the dynamical interactions between peptide P62 and a structural model of the λ light chain in systemic amyloidosis. We found that P62 only binds to the canonical interface of the fibril where the peptide inserts into the fibril groove and its two termini are more mobile than the helix core. Our results also revealed an important role of the lysine residues of P62 in the binding process by forming initial contacts with aspartic acids on the fibril surface. Collectively, our computational study provided molecular-level insights into the binding mechanism between an amyloid fibril model and peptide P62, which could lay a foundation for rational design of peptides for improved amyloid diagnosis and immunotherapy.
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Proteínas Amiloidogénicas/química , Péptidos/química , Humanos , Simulación de Dinámica Molecular , Péptidos/síntesis química , Unión Proteica , Conformación ProteicaRESUMEN
Peptide p5R is a synthetic, polybasic, heparin-binding peptide that preferentially reacts with amyloid deposits in vivo and in tissue sections. Basic fibroblast growth factor (bFGF1) similarly interacts with heparin-like molecules, notably heparan sulfate proteoglycans (HSPG), in the extracellular matrix and on cell surfaces. The aim of this study was to compare the biodistribution of p5R and bFGF in healthy mice as well as those with systemic inflammation-associated amyloidosis (AA), which contains HSPG, by using SPECT/CT imaging, tissue biodistribution measurements and micro-autoradiography. Although both proteins are known to bind heparan sulfate, their biodistribution was remarkably different in the healthy and diseased animals. Imaging revealed uptake of both radiolabeled proteins in the liver, spleen, and kidneys of mice with amyloidosis; however, 125I-bFGF, but not 125I-p5R, was observed in normal tissue at sites of HSPG expression, including the hepatic and splenic sinusoids and renal glomerulae. Microautoradiography demonstrated that while p5R bound exclusively to amyloid deposits in the spleen and liver of AA mice, bFGF had a broader binding pattern. Consequently, even though bFGF and p5R both interact with heparan sulfate moieties, p5R binding was restricted to HSPG in amyloid deposits and did not bind HSPG in healthy tissues, whereas bFGF preferentially reacted with HSPG in normal tissue. The data suggest that peptide p5R selectively binds HSPG in amyloid and that the HSPG in healthy tissue, recognized by bFGF, is not targeted by the peptide.
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Amiloide/metabolismo , Amiloidosis/metabolismo , Factor 2 de Crecimiento de Fibroblastos/metabolismo , Heparina/metabolismo , Péptidos/metabolismo , Amiloidosis/diagnóstico por imagen , Animales , Autorradiografía/métodos , Factor 2 de Crecimiento de Fibroblastos/química , Heparina/química , Radioisótopos de Yodo/metabolismo , Radioisótopos de Yodo/farmacocinética , Hígado/metabolismo , Ratones Endogámicos BALB C , Ratones Transgénicos , Estructura Molecular , Péptidos/química , Dominios Proteicos , Tomografía Computarizada por Tomografía Computarizada de Emisión de Fotón Único/métodos , Bazo/metabolismo , Distribución TisularRESUMEN
Human cytomegalovirus (HCMV) is a ubiquitous betaherpesvirus that can cause severe disease following in utero exposure, during primary infection, or latent virus reactivation in immunocompromised populations. These complications lead to a 1- to 2-billion-dollar economic burden, making vaccine development and/or alternative treatments a high priority. Current treatments for HCMV include nucleoside analogues such as ganciclovir (GCV), foscarnet, and cidofovir. Recently, letermovir, a terminase complex inhibitor, was approved for prophylaxis after stem cell transplantation. These treatments have unwanted side effects, and HCMV is becoming resistant to them. Therefore, we sought to develop an alternative treatment that targets a different stage in viral infection. Currently, small antiviral peptides are being investigated as anti-influenza and anti-HIV treatments. We have developed heparan sulfate-binding peptides as tools for preventing CMV infections. These peptides are highly effective at stopping infection of fibroblasts with in vitro-derived HCMV and murine cytomegalovirus (MCMV). However, they do not prevent MCMV infection in vivo Interestingly, these peptides inhibit infectivity of in vivo-derived CMVs, albeit not as well as tissue culture-grown CMVs. We further demonstrate that this class of heparan sulfate-binding peptides is incapable of inhibiting MCMV cell-to-cell spread, which is independent of heparan sulfate usage. These data indicate that inhibition of CMV infection can be achieved using synthetic polybasic peptides, but cell-to-cell spread and in vivo-grown CMVs require further investigation to design appropriate anti-CMV peptides.IMPORTANCE In the absence of an effective vaccine to prevent HCMV infections, alternative interventions must be developed. Prevention of viral entry into susceptible cells is an attractive alternative strategy. Here we report that heparan sulfate-binding peptides effectively inhibit entry into fibroblasts of in vitro-derived CMVs and partially inhibit in vivo-derived CMVs. This includes the inhibition of urine-derived HCMV (uCMV), which is highly resistant to antibody neutralization. While these antiviral peptides are highly effective at inhibiting cell-free virus, they do not inhibit MCMV cell-to-cell spread. This underscores the need to understand the mechanism of cell-to-cell spread and differences between in vivo-derived versus in vitro-derived CMV entry to effectively prevent CMV's spread.
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Antivirales/farmacología , Infecciones por Citomegalovirus/prevención & control , Citomegalovirus/efectos de los fármacos , Citomegalovirus/fisiología , Péptidos/farmacología , Animales , Células Cultivadas , Infecciones por Citomegalovirus/tratamiento farmacológico , Modelos Animales de Enfermedad , Fibroblastos/virología , Heparitina Sulfato/metabolismo , Humanos , Ratones , Ratones Endogámicos BALB C , Muromegalovirus/efectos de los fármacos , Internalización del Virus/efectos de los fármacos , Replicación Viral/efectos de los fármacosRESUMEN
pH-responsive peptides are promising therapeutic molecules that can specifically target the plasma membrane in the acidified extracellular medium that bathes cells in tumors. We designed the acidity-triggered rational membrane (ATRAM) peptide to have a pH-responsive membrane interaction. At physiological pH, ATRAM binds to the membrane surface in a largely unstructured conformation, while in acidic conditions it inserts into lipid bilayers forming a transmembrane helix. However, the molecular mechanism ATRAM uses to target and insert into tumor cells remains poorly understood. Here, we determined that ATRAM inserts into cancer cells with a preferential membrane orientation, where the C-terminus of the peptide traverses the plasma membrane and explores the cytoplasm. Using biophysical techniques, we determined that the membrane interaction of ATRAM is contingent on the concentration of the peptide. Kinetic studies showed that membrane insertion occurs in at least three steps, where only the first step was affected by the membrane density of ATRAM. These observations, combined with membrane binding and leakage data, indicate that the interaction of ATRAM with lipid membranes is dependent on its oligomerization state. SPECT/CT imaging in mice revealed that ATRAM accumulates in the blood pool, where it has a prolonged circulation time (> 4â¯h). Since fast peptide clearance and degradation in circulation are major problems for clinical development, we studied the mechanism ATRAM uses to remain in the blood stream. Using binding and transfer assays, we determined that ATRAM binds reversibly to human serum albumin. We propose that ATRAM uses albumin as a carrier in the blood stream to evade clearance and proteolysis before interacting with the plasma membrane of cancer cells. We also show that ATRAM is able to be deliver liposomes to cells in a pH dependent way. Our data highlight the potential of ATRAM as a specific therapeutic agent for diseases that lead to acidic tissues, including cancer.
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Membrana Celular/metabolismo , Péptidos/metabolismo , Albúmina Sérica Humana/metabolismo , Animales , Neoplasias de la Mama/metabolismo , Femenino , Humanos , Concentración de Iones de Hidrógeno , Membrana Dobles de Lípidos/metabolismo , Liposomas , Células MCF-7 , Lípidos de la Membrana/metabolismo , Proteínas de la Membrana/metabolismo , Ratones , Ratones Endogámicos BALB CRESUMEN
Light chain-associated amyloidosis is characterized by the extracellular deposition of amyloid fibrils in abdominothoracic organs, skin, soft tissue, and peripheral nerves. Phagocytic cells of the innate immune system appear to be ineffective at clearing the material; however, human light chain amyloid extract, injected subcutaneously into mice, is rapidly cleared in a process that requires neutrophil activity. To better elucidate the phagocytosis of light chain fibrils, a potential method of cell-mediated dissolution, amyloid-like fibrils were labeled with the pH-sensitive dye pHrodo red and a near infrared fluorophore. After injecting this material subcutaneously in mice, optical imaging was used to quantitatively monitor phagocytosis and dissolution of fibrils concurrently. Histologic evaluation of the residual fibril masses revealed the presence of CD68+, F4/80+, ionized calcium binding adaptor molecule 1- macrophages containing Congo red-stained fibrils as well as neutrophil-associated proteins with no evidence of intact neutrophils. These data suggest an early infiltration of neutrophils, followed by extensive phagocytosis of the light chain fibrils by macrophages, leading to dissolution of the mass. Optical imaging of this novel murine model, coupled with histologic evaluation, can be used to study the cellular mechanisms underlying dissolution of synthetic amyloid-like fibrils and human amyloid extracts. In addition, it may serve as a test bed to evaluate investigational opsonizing agents that might serve as therapeutic agents for light chain-associated amyloidosis.
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Amiloide/fisiología , Amiloidosis/patología , Macrófagos/fisiología , Imagen Óptica/métodos , Fagocitosis , Animales , Femenino , Macrófagos/citología , RatonesRESUMEN
3'-deoxy-3'-[18 F]fluorothymidine (18 FLT) is a radiopharmaceutical tracer used with positron emission tomography (PET), often in combination with computed tomography (CT), to image DNA synthesis, and thus, cellular proliferation. Characteristic accumulation of the tracer within haematopoietic bone marrow provides a noninvasive means to assess marrow activity and distribution throughout the living animal. The present study utilizes three-dimensional analysis of 18 FLT-PET/CT scans to quantify the relative skeletal distribution of active marrow by anatomic site in the dog. Scans were performed on six healthy, adult (3-6 years of age), mixed-breed dogs using a commercially available PET/CT scanner consisting of a 64-slice helical CT scanner combined with an integrated four ring, high-resolution LSO PET scanner. Regions of interest encompassing 11 separate skeletal regions (skull, cervical vertebral column, thoracic vertebral column, lumbar vertebral column, sacrum, ribs, sternum, scapulae, proximal humeri, ossa coxarum, and proximal femora) were manually drawn based on CT images and thresholded by standardized uptake value to delineate bone marrow activity. Activity within each skeletal region was then divided by the total skeletal activity to derive the per cent of overall marrow activity within an individual site. The majority of proliferative marrow was located within the vertebral column. Of the sites traditionally accessed clinically for marrow sampling, the proximal humerus contained the largest percentage, followed by the ossa coxarum, proximal femur, and sternum, respectively. This information may be used to guide selection of traditional marrow sampling sites as well as inform efforts to spare important sites of haematopoiesis in radiation therapy planning.
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Médula Ósea/fisiología , Huesos/anatomía & histología , Tomografía Computarizada por Tomografía de Emisión de Positrones/veterinaria , Animales , Perros , Femenino , Masculino , Radiofármacos , Timidina/análogos & derivadosRESUMEN
Amyloidosis is a malignant pathology associated with the formation of proteinaceous amyloid fibrils that deposit in organs and tissues, leading to dysfunction and severe morbidity. More than 25 proteins have been identified as components of amyloid, but the most common form of systemic amyloidosis is associated with the deposition of amyloid composed of Ig light chains (AL). Clinical management of amyloidosis focuses on reducing synthesis of the amyloid precursor protein. However, recently, passive immunotherapy using amyloid fibril-reactive antibodies, such as 11-1F4, to remove amyloid from organs has been shown to be effective at restoring organ function in patients with AL amyloidosis. However, 11-1F4 does not bind amyloid in all AL patients, as evidenced by PET/CT imaging, nor does it efficiently bind the many other forms of amyloid. To enhance the reactivity and expand the utility of the 11-1F4 mAb as an amyloid immunotherapeutic, we have developed a pretargeting "peptope" comprising a multiamyloid-reactive peptide, p5+14, fused to a high-affinity peptide epitope recognized by 11-1F4. The peptope, known as p66, bound the 11-1F4 mAb in vitro with subnanomolar efficiency, exhibited multiamyloid reactivity in vitro and, using tissue biodistribution and SPECT imaging, colocalized with amyloid deposits in a mouse model of systemic serum amyloid A amyloidosis. Pretreatment with the peptope induced 11-1F4 mAb accumulation in serum amyloid A deposits in vivo and enhanced 11-1F4-mediated dissolution of a human AL amyloid extract implanted in mice.
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Amiloidosis/metabolismo , Amiloidosis/terapia , Anticuerpos Monoclonales/fisiología , Amiloide/metabolismo , Proteínas Amiloidogénicas/metabolismo , Animales , Anticuerpos Biespecíficos/inmunología , Anticuerpos Monoclonales/inmunología , Cadáver , Epítopos/metabolismo , Humanos , Cadenas Ligeras de Inmunoglobulina/inmunología , Ratones , Péptidos/metabolismo , Tomografía Computarizada por Tomografía de Emisión de Positrones , Unión Proteica , Proteína Amiloide A Sérica/metabolismo , Distribución Tisular , Resultado del TratamientoRESUMEN
INTRODUCTION: Multiple myeloma (MM) and light chain monoclonal gammopathy of undetermined significance (LCMGUS) are plasma cell disorders associated with the secretion of monoclonal free light-chain (LC) proteins. Due to the high concentrations of LC in circulation, both of these populations are at risk for developing LC-associated amyloidosis (AL) - a protein misfolding disease characterized by the deposition of LC protein fibrils in organs and tissues, leading to dysfunction and significant morbidity. At present, accurate identification of subjects at risk for developing amyloidosis is not possible, but with the advent of novel, amyloid-targeted therapies, identification of pre-symptomatic individuals is of clinical import. METHODS: To address this, a competition assay has been developed to discern LC proteins with enhanced amyloidogenic potential. Numerous factors that may influence the efficacy of the assay have been evaluated to yield optimal conditions. RESULTS: Using a panel of nine patient-derived LC, we have demonstrated that amyloid-associated LC inhibited the recruitment of a biotinyl-λ6 variable domain by homologous amyloid-like fibrils significantly more than MM LC (p < .01). CONCLUSION: The assay accurately discriminated AL from MM patient populations, suggesting that it may aid in the identification of patients with monoclonal gammopathies who have an increased risk of developing amyloidosis.
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Bioensayo/métodos , Amiloidosis de Cadenas Ligeras de las Inmunoglobulinas/diagnóstico , Mieloma Múltiple/diagnóstico , Paraproteinemias/diagnóstico , Proteínas Amiloidogénicas/metabolismo , Humanos , Amiloidosis de Cadenas Ligeras de las Inmunoglobulinas/metabolismo , Mieloma Múltiple/metabolismo , Paraproteinemias/metabolismoRESUMEN
BACKGROUND: Systemic amyloidoses comprise diseases characterized by the deposition of proteinaceous material known as amyloid. Currently, without performing multiple biopsies, there is no way to ascertain the extent of amyloid deposition in patients-a critical piece of information that informs prognosis and therapeutic strategies. We have developed pan-amyloid-targeting peptides for imaging amyloid and recently have adapted these for use as pre-targeting agents in conjunction with immunotherapy. Incorporation of D-amino acids in these peptides may enhance serum half-life, which is an important characteristic of effective peptide therapeutics. Herein, we assess the effects of partial incorporation of D-amino acids into the amyloidophilic peptide p5 on in vivo amyloid reactivity. METHODS: Peptides, referred to as AQAp5 (d) , aqap5, and AQAp5, were radiolabeled with iodine-125 and the tissue biodistribution (% injected dose/gram) measured in healthy mice at multiple time points post-injection. Microscopic distribution of the peptides was further visualized using microautoradiography (ARG). Peptides aqap5 and AQAp5 were injected into healthy and amyloid-laden mice and evaluated by using SPECT/CT imaging at 1, 4 and 24 h post injection. RESULTS: Biodistribution data and ARG revealed persistent retention of [125I]AQAp5 (d) in the liver and kidneys of healthy mice for at least 24 h. In contrast, peptides [125I]aqap5 and [125I]AQAp5 did not bind these organs and was significantly lower than [125I]AQAp5 (d) at 24 h post injection (p < 0.0001). SPECT/CT imaging of amyloid-laden mice revealed accumulation of both [125I]aqap5 and [125I]AQAp5 in amyloid-affected organs; whereas, in healthy mice, [125I]aqap5 was observed in the kidneys and liver at early time points, and free radioiodide liberated during catabolism of [125I]AQAp5 was seen in the stomach and thyroid. Autoradiography confirmed that both [125I]aqap5 and [125I]AQAp5 peptides specifically bound amyloid with no off-target binding to healthy organs. CONCLUSION: Incorporation of D-amino acids in amyloid-binding regions of amyloidophilic peptides resulted in off-target binding; however, N-terminus placement retained amyloid-specificity and evasion of deiodinases. Peptide aqap5, or similar reagents, may prove useful in novel immunotherapy strategies as well as for imaging renal, gastric and pancreatic amyloidosis.
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Aminoácidos/metabolismo , Amiloide/metabolismo , Péptidos/metabolismo , Secuencia de Aminoácidos , Animales , Autorradiografía , Humanos , Ratones Transgénicos , Péptidos/química , Estructura Secundaria de Proteína , Distribución Tisular , Tomografía Computarizada de Emisión de Fotón Único , Tomografía Computarizada por Rayos XRESUMEN
There is a continuing need for therapeutic interventions for patients with the protein misfolding disorders that result in systemic amyloidosis. Recently, specific antibodies have been employed to treat AL amyloidosis by opsonizing tissue amyloid deposits thereby inducing cell-mediated dissolution and organ improvement. To develop a pan-amyloid therapeutic agent, we have produced an Fc-fusion product incorporating a peptide, p5, which binds many if not all forms of amyloid. This protein, designated Fcp5, expressed in mammalian cells, forms the desired bivalent dimer structure and retains pan-amyloid reactivity similar to the p5 peptide as measured by immunosorbent assays, immunohistochemistry, surface plasmon resonance, and pulldown assays using radioiodinated Fcp5. Additionally, Fcp5 was capable of opsonizing amyloid fibrils in vitro using a pH-sensitive fluorescence assay of phagocytosis. In mice,125 I-labeled Fcp5 exhibited an extended serum circulation time, relative to the p5 peptide. It specifically bound AA amyloid deposits in diseased mice, as evidenced by biodistribution and microautoradiographic methods, which coincided with an increase in active, Iba-1-positive macrophages in the liver at 48 h postinjection of Fcp5. In healthy mice, no specific tissue accumulation was observed. The data indicate that polybasic, pan-amyloid-targeting peptides, in the context of an Fc fusion, can yield amyloid reactive, opsonizing reagents that may serve as next-generation immunotherapeutics.
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The amyloidoses are a complex group of disorders characterized by the deposition of proteinaceous amyloid fibrils in vital organs. The deposits are nonimmunogenic and may be composed of one of more than 35 proteins. We have developed a two-stage immunotherapeutic approach using peptides that recognize most, if not all, amyloid deposits to facilitate amyloid clearance. In the first embodiment, we have developed a bifunctional peptope to enhance and expand the utility of currently available antibodies. In the second, we have generated peptide-reactive antibodies that can be targeted to the amyloid deposits by peptides thereby providing alternative reagents for immunotherapy of amyloidosis. These technologies provide tools for treating the many forms of amyloid disease, restoring organ function and enhancing patient survival.
Asunto(s)
Inmunoterapia , Amiloide , Amiloidosis , Humanos , Mieloma Múltiple , PéptidosRESUMEN
Amyloidosis is associated with a number of rare diseases and is characterized by the deposition, in abdominothoracic organs and peripheral nerves, of extracellular protein fibrils, which leads to dysfunction and severe morbidity. Effective clinical evaluation and management of patients with systemic amyloidosis are hampered by the lack of a noninvasive, quantitative method for detecting whole-body amyloid load. We have used a battery of assays including dual-energy SPECT imaging and comparative effectiveness studies in support of translation of a synthetic polybasic peptide, p5+14, as a novel radiotracer for visualization of amyloidosis by molecular imaging. These data provide support for a phase 1 positron emission tomography/computed tomography imaging trial of this reagent, labeled with iodine-124, in patients with all forms of systemic amyloidosis.