Discrete binding patterns of two heparin-reactive proteins, basic fibroblast growth factor and peptide p5R, in amyloid-laden and healthy mice.

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.

Computational investigation of the binding of a designed peptide to λ light chain amyloid fibril.

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.

Bifunctional amyloid-reactive peptide promotes binding of antibody 11-1F4 to diverse amyloid types and enhances therapeutic efficacy.

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.

Macrophage-Mediated Phagocytosis and Dissolution of Amyloid-Like Fibrils in Mice, Monitored by Optical Imaging.

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.

Dual-Energy SPECT and the Development of Peptide p5+14 for Imaging Amyloidosis.

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.

Evaluation of the effect of D-amino acid incorporation into amyloid-reactive peptides.

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.

Preclinical Validation of the Heparin-Reactive Peptide p5+14 as a Molecular Imaging Agent for Visceral Amyloidosis.

Amyloid is a complex pathologic matrix comprised principally of paracrystalline protein fibrils and heparan sulfate proteoglycans. Systemic amyloid diseases are rare, thus, routine diagnosis is often challenging. The glycosaminoglycans ubiquitously present in amyloid deposits are biochemically and electrochemically distinct from those found in the healthy tissues due to the high degree of sulfation. We have exploited this unique property and evaluated heparin-reactive peptides, such as p5+14, as novel agents for specifically targeting and imaging amyloid. Herein, we demonstrate that radiolabeled p5+14 effectively bound murine AA amyloid in vivo by using molecular imaging. Biotinylated peptide also reacted with the major forms of human amyloid in tissue sections as evidenced immunohistochemically. Furthermore, we have demonstrated that the peptide also binds synthetic amyloid fibrils that lack glycosaminoglycans implying that the dense anionic motif present on heparin is mimicked by the amyloid protein fibril itself. These biochemical and functional data support the translation of radiolabeled peptide p5+14 for the clinical imaging of amyloid in patients.

In vivo molecular imaging of peripheral amyloidosis using heparin-binding peptides.

Heparan sulfate proteoglycans (HSPGs) are ubiquitous components of pathologic amyloid deposits in the organs of patients with disorders such as Alzheimer's disease or systemic light chain (AL) or reactive (AA) amyloidosis. Molecular imaging methods for early detection are limited and generally unavailable outside the United Kingdom. Therefore, there is an urgent need to develop novel, specific amyloidophilic radiotracers for imaging to assist in diagnosis, prognostication, and monitoring response to therapy. Amyloid-associated HSPG can be differentiated from HSPG found in surrounding healthy cells and tissues by the preferential binding of certain HS-reactive single chain variable fragments and therefore, represents a biomarker that can be targeted specifically with appropriate reagents. Using a murine model of AA amyloidosis, we have examined the in vivo amyloid reactivity of seven heparin-binding peptides by using single photon emission and X-ray computed tomographic imaging, microautoradiography, and tissue biodistribution measurements. All of the peptides bound amyloid deposits within 1 h post-injection, but the extent of the reactivity differed widely, which was evidenced by image quality and grain density in autoradiographs. One radiolabeled peptide bound specifically to murine AA amyloid in the liver, spleen, kidney, adrenal, heart, and pancreas with such avidity that it was observed in single photon emission tomography images as late as 24 h post-injection. In addition, a biotinylated form of this peptide was shown histochemically to bind human AA, ALκ, ALλ, transthyretin amyloidosis (ATTR), and Aß amyloid deposits in tissue sections. These basic heparin-binding peptides recognize murine and human amyloid deposits in both in vivo and ex vivo tissues and therefore, have potential as radiotracers for the noninvasive molecular imaging of amyloid deposits in situ.

Preclinical evaluation of Tc-99m p5+14 peptide for SPECT detection of cardiac amyloidosis.

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.

Peptide p5 binds both heparinase-sensitive glycosaminoglycans and fibrils in patient-derived AL amyloid extracts.

In previously published work, we have described heparin-binding synthetic peptides that preferentially recognize amyloid deposits in a mouse model of reactive systemic (AA) amyloidosis and can be imaged by using positron and single photon emission tomographic imaging. We wanted to extend these findings to the most common form of visceral amyloidosis, namely light chain (AL); however, there are no robust experimental animal models of AL amyloidosis. To further define the binding of the lead peptide, p5, to AL amyloid, we characterized the reactivity in vitro of p5 with in situ and patient-derived AL amyloid extracts which contain both hypersulfated heparan sulfate proteoglycans as well as amyloid fibrils. Histochemical staining demonstrated that the peptide specifically localized with tissue-associated AL amyloid deposits. Although we anticipated that p5 would undergo electrostatic interactions with the amyloid-associated glycosaminoglycans expressing heparin-like side chains, no significant correlation between peptide binding and glycosaminoglycan content within amyloid extracts was observed. In contrast, following heparinase I treatment, although overall binding was reduced, a positive correlation between peptide binding and amyloid fibril content became evident. This interaction was further confirmed using synthetic light chain fibrils that contain no carbohydrates. These data suggest that p5 can bind to both the sulfated glycosaminoglycans and protein fibril components of AL amyloid. Understanding these complex electrostatic interactions will aid in the optimization of synthetic peptides for use as amyloid imaging agents and potentially as therapeutics for the treatment of amyloid diseases.

Whole-body biodistribution of 3'-deoxy-3'-[(18) f]fluorothymidine ((18) FLT) in healthy adult cats.

Positron emission tomography/computed tomography (PET/CT) utilizing 3'-deoxy-3'-[(18) F]fluorothymidine ((18) FLT), a proliferation tracer, has been found to be a useful tool for characterizing neoplastic diseases and bone marrow function in humans. As PET and PET/CT imaging become increasingly available in veterinary medicine, knowledge of radiopharmaceutical biodistribution in veterinary species is needed for lesion interpretation in the clinical setting. The purpose of this study was to describe the normal biodistribution of (18) FLT in adult domestic cats. Imaging of six healthy young adult castrated male cats was performed using a commercially available PET/CT scanner consisting of a 64-slice helical CT scanner with an integrated whole-body, high-resolution lutetium oxy-orthosilicate (LSO) PET scanner. Cats were sedated and injected intravenously with 108.60 ± 2.09 (mean ± SD) MBq of (18) FLT (greater than 99% radiochemical purity by high-performance liquid chromatography). Imaging was performed in sternal recumbency under general anesthesia. Static images utilizing multiple bed positions were acquired 80.83 ± 7.52 (mean ± SD) minutes post-injection. Regions of interest were manually drawn over major parenchymal organs and selected areas of bone marrow and increased tracer uptake. Standardized uptake values were calculated. Notable areas of uptake included hematopoietic bone marrow, intestinal tract, and the urinary and hepatobiliary systems. No appreciable uptake was observed within brain, lung, myocardium, spleen, or skeletal muscle. Findings from this study can be used as baseline data for future studies of diseases in cats.

Collagen inhibits phagocytosis of amyloid in vitro and in vivo and may act as a 'don't eat me' signal.

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.

Cardiac Amyloid Detection by PET/CT Imaging of Iodine (124I) Evuzamitide (124I-p5+14): A Phase 1/2 Study.

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.

Development and characterization of a prototypic pan-amyloid clearing agent - a novel murine peptide-immunoglobulin fusion.

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.

Clinical Confirmation of Pan-Amyloid Reactivity of Radioiodinated Peptide 124I-p5+14 (AT-01) in Patients with Diverse Types of Systemic Amyloidosis Demonstrated by PET/CT Imaging.

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.

Radioimmunodetection of amyloid deposits in patients with AL amyloidosis.

Care of patients with AL amyloidosis currently is limited by the lack of objective means to document disease extent, as well as therapeutic options that expedite removal of pathologic deposits. To address these issues, we have initiated a Phase I Exploratory IND study to determine the biodistribution of the fibril-reactive, amyloidolytic murine IgG1 mAb 11-1F4 labeled with I-124. Patients were infused with less than 1 mg (∼ 74 MBq) of GMP-grade antibody and imaged by PET/CT scan 48 and 120 hours later. Among 9 of 18 subjects, there was striking uptake of the reagent in liver, lymph nodes, bone marrow, intestine, or, unexpectedly, spleen (but not kidneys or heart). Generally, positive or negative results correlated with those obtained immunohistochemically using diagnostic tissue biopsy specimens. Based on these findings, we posit that (124)I-mAb m11-1F4 can be used to identify AL candidates for passive immunotherapy using the chimeric form of the antibody.

First in Human Evaluation and Dosimetry Calculations for Peptide 124I-p5+14-a Novel Radiotracer for the Detection of Systemic Amyloidosis Using PET/CT Imaging.

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.

The heparan sulfate motif (GlcNS6S-IdoA2S)3, common in heparin, has a strict topography and is involved in cell behavior and disease.

Heparan sulfate (HS) is a structurally complex polysaccharide that interacts with a broad spectrum of extracellular effector ligands and thereby is thought to regulate a diverse array of biologic processes. The specificity of HS-ligand interactions is determined by the arrangement of sulfate groups on HS, which creates distinct binding motifs. Biologically important HS motifs are expected to exhibit regulated expression, yet there is a profound lack of tools to identify such motifs; consequently, little is known of their structures and functions. We have identified a novel phage display-derived antibody (NS4F5) that recognizes a highly regulated HS motif (HS(NS4F5)), which we have rigorously identified as (GlcNS6S-IdoA2S)(3). HS(NS4F5) exhibits a restricted expression in healthy adult tissues. Blocking HS(NS4F5) on cells in culture resulted in reduced proliferation and enhanced sensitivity to apoptosis. HS(NS4F5) is up-regulated in tumor endothelial cells, consistent with a role in endothelial cell activation. Indeed, TNF-α stimulated endothelial expression of HS(NS4F5), which contributed to leukocyte adhesion. In a mouse model of severe systemic amyloid protein A amyloidosis, HS(NS4F5) was expressed within amyloid deposits, which were successfully detected by microSPECT imaging using NS4F5 as a molecularly targeted probe. Combined, our results demonstrate that NS4F5 is a powerful tool for elucidating the biological function of HS(NS4F5) and can be exploited as a probe to detect novel polysaccharide biomarkers of disease processes.

LaPO4 nanoparticles doped with actinium-225 that partially sequester daughter radionuclides.

Nanoscale materials have been envisioned as carriers for various therapeutic drugs, including radioisotopes. Inorganic nanoparticles (NPs) are particularly appealing vehicles for targeted radiotherapy because they can package several radioactive atoms into a single carrier and can potentially retain daughter radioisotopes produced by in vivo generators such as actinium-225 ((225)Ac, t(1/2) = 10 d). Decay of this radioisotope to stable bismuth-209 proceeds through a chain of short-lived daughters accompanied by the emission of four α-particles that release >27 MeV of energy. The challenge in realizing the enhanced cytotoxic potential of in vivo generators lies in retaining the daughter nuclei at the therapy site. When (225)Ac is attached to targeting agents via standard chelate conjugation methods, all of the daughter radionuclides are released after the initial α-decay occurs. In this work, (225)Ac was incorporated into lanthanum phosphate NPs to determine whether the radioisotope and its daughters would be retained within the dense mineral lattice. Further, the (225)Ac-doped NPs were conjugated to the monoclonal antibody mAb 201B, which targets mouse lung endothelium through the vasculature, to ascertain the targeting efficacy and in vivo retention of radioisotopes. Standard biodistribution techniques and microSPECT/CT imaging of (225)Ac as well as the daughter radioisotopes showed that the NPs accumulated rapidly in mouse lung after intravenous injection. By showing that excess, competing, uncoupled antibodies or NPs coupled to control mAbs are deposited primarily in the liver and spleen, specific targeting of NP-mAb 201B conjugates was demonstrated. Biodistribution analysis showed that ∼30% of the total injected dose of La((225)Ac)PO(4) NPs accumulated in mouse lungs 1 h postinjection, yielding a value of % ID/g >200. Furthermore, after 24 h, 80% of the (213)Bi daughter produced from (225)Ac decay was retained within the target organ and (213)Bi retention increased to ∼87% at 120 h. In vitro analyses, conducted over a 1 month interval, demonstrated that ∼50% of the daughters were retained within the La((225)Ac)PO(4) NPs at any point over that time frame. Although most of the γ-rays from radionuclides in the (225)Ac decay chain are too energetic to be captured efficiently by SPECT detectors, appropriate energy windows were found that provided dramatic microSPECT images of the NP distribution in vivo. We conclude that La((225)Ac)PO(4)-mAb 201B conjugates can be targeted efficiently to mouse lung while partially retaining daughter products and that targeting can be monitored by biodistribution techniques and microSPECT imaging.

Discrimination of lesion removal of N-methylpurine-DNA glycosylase revealed by a potent neutralizing monoclonal antibody.

N-Methylpurine-DNA glycosylase (MPG), a ubiquitous DNA repair enzyme, initiates excision repair of several N-alkylpurine adducts, induced by alkylating chemotherapeutics, and deaminated and lipid peroxidation-induced purine adducts. We have generated monoclonal antibodies (moAbs) against human MPG. Twelve independent hybridoma clones were characterized, which, except 520-16A, are identical based on epitope exclusion assay. Four moAbs, including 520-2A, 520-3A, 520-16A, and 520-26A, have high affinity (K(D) approximately 0.3-1.6nM), and their subtypes were IgG(2a), IgG(1), IgG(2a), and IgG(2b), respectively. moAb 520-3A recognizes the sequence (52)AQAPCPRERCLGPP(66)T, an epitope exclusively present in the N-terminal extension of human MPG. We found that moAb 520-3A significantly inhibited MPG's enzymatic activity towards different substrates, such as hypoxanthine, 1,N(6)ethenoadenine and methylated bases, which represent different classes of DNA damage, however, with different efficiencies. Real-time binding experiments using surface plasmon resonance (SPR) spectroscopy showed that the pronounced inhibition of activity was not in the substrate-binding step. Single turnover kinetics (STO) revealed that the inhibition was at the catalytic step. Since we found that this antibody has an epitope in the N-terminal tail, the latter appears to have an important role in substrate discrimination, however, with a differential effect on different substrates.