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.

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.

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.

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.

A functional assay to identify amyloidogenic light chains.

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.

Pretargeting immunotherapy: a novel treatment approach for systemic amyloidosis.

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.

Kinetic stability and sequence/structure studies of urine-derived Bence-Jones proteins from multiple myeloma and light chain amyloidosis patients.

It is now accepted that the ability of a protein to form amyloid fibrils could be associated both kinetic and thermodynamic protein folding parameters. A recent study from our laboratory using recombinant full-length (encompassing the variable and constant domain) immunoglobulin light chains found a strong kinetic control of the protein unfolding for these proteins. In this study, we are extending our analysis by using urine-derived Bence Jones proteins (BJPs) from five patients with light chain (AL) amyloidosis and four patients with multiple myeloma (MM). We observed lower stability in κ proteins compared to λ proteins (for both MM and AL proteins) in agreement with previous studies. The kinetic component of protein stability is not a universal feature of BJPs and the hysteresis observed during refolding reactions could be attributed to the inability of the protein to refold all domains. The most stable proteins exhibited 3-state unfolding transitions. While these proteins do not refold reversibly, partial refolding shows 2-state partial refolding transitions, suggesting that one of the domains (possibly the variable domain) does not refold completely. Sequences were aligned with their respective germlines and the location and nature of the mutations were analyzed. The location of the mutations were analyzed and compared with the stability and amyloidogenic properties for the proteins in this study, increasing our understanding of light chain unfolding and amyloidogenic potential.

A Peptide-Fc Opsonin with Pan-Amyloid Reactivity.

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.