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.

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.

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.

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.

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.

The clinical correlation of phosphatase and tensin homolog on chromosome 10, phosphorylation of AKT to an activated state, and odontogenic ameloblast-associated protein in gastrointestinal stromal tumors.

BACKGROUND: Approximately 15% of gastrointestinal stromal tumors (GISTs) will not respond to tyrosine kinase inhibitors and drug resistance can develop over time. For refractory tumors, additional therapies are needed. Odontogenic ameloblast-associated protein (ODAM) is expressed in some epithelial malignancies and can correlate with clinical outcomes. This study evaluated ODAM and its relationship to phosphatase and tensin homolog on chromosome 10 (PTEN) and phosphorylation of AKT to an activated state (pAKT) in GISTs. MATERIALS AND METHODS: Ninety-five distinct tumor specimens from 79 patients were identified. Morphologic features and clinical data were recorded for all tumors. Risk of recurrence was calculated using the Memorial Sloan-Kettering nomogram. Immunohistochemistry was performed using antibodies to ODAM, PTEN, and pAKT. Immunoreactivity was assessed for both cytoplasmic and nuclear expression. Staining patterns were correlated with clinical outcomes. RESULTS: Increasing cytoplasmic ODAM staining correlated with a lower recurrence score (P = 0.002), a lower mitotic rate (P = 0.0001), and smaller tumor size (P = 0.038). Increasing pAKT cytoplasmic staining correlated with a higher recurrence score (P = 0.037) and a higher mitotic rate (P = 0.036). ODAM and pAKT expression in the nucleus was associated with tumor origin. PTEN nuclear expression increased with increasing mitotic rate. pAKT expression increased in the cytoplasm and nucleus in high-risk tumors. CONCLUSIONS: Risk of recurrence correlated with cytoplasmic expression of ODAM and pAKT, whereas nuclear expression did not predict recurrence. The staining pattern for ODAM and pAKT in the cytoplasm may further clarify the risk of recurrence beyond the available nomograms. The increased expression of pAKT in the cytoplasm and nucleus of high-risk tumors suggests a potential target for systemic therapy.

Light Chain Amyloid Fibrils Cause Metabolic Dysfunction in Human Cardiomyocytes.

Light chain (AL) amyloidosis is the most common form of systemic amyloid disease, and cardiomyopathy is a dire consequence, resulting in an extremely poor prognosis. AL is characterized by the production of monoclonal free light chains that deposit as amyloid fibrils principally in the heart, liver, and kidneys causing organ dysfunction. We have studied the effects of amyloid fibrils, produced from recombinant λ6 light chain variable domains, on metabolic activity of human cardiomyocytes. The data indicate that fibrils at 0.1 µM, but not monomer, significantly decrease the enzymatic activity of cellular NAD(P)H-dependent oxidoreductase, without causing significant cell death. The presence of amyloid fibrils did not affect ATP levels; however, oxygen consumption was increased and reactive oxygen species were detected. Confocal fluorescence microscopy showed that fibrils bound to and remained at the cell surface with little fibril internalization. These data indicate that AL amyloid fibrils severely impair cardiomyocyte metabolism in a dose dependent manner. These data suggest that effective therapeutic intervention for these patients should include methods for removing potentially toxic amyloid fibrils.

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.

A human monoclonal IgG that binds aß assemblies and diverse amyloids exhibits anti-amyloid activities in vitro and in vivo.

Alzheimer's disease (AD) and familial Danish dementia (FDD) are degenerative neurological diseases characterized by amyloid pathology. Normal human sera contain IgG antibodies that specifically bind diverse preamyloid and amyloid proteins and have shown therapeutic potential in vitro and in vivo. We cloned one of these antibodies, 3H3, from memory B cells of a healthy individual using a hybridoma method. 3H3 is an affinity-matured IgG that binds a pan-amyloid epitope, recognizing both Aß and λ Ig light chain (LC) amyloids, which are associated with AD and primary amyloidosis, respectively. The pan-amyloid-binding properties of 3H3 were demonstrated using ELISA, immunohistochemical studies, and competition binding assays. Functional studies showed that 3H3 inhibits both Aß and LC amyloid formation in vitro and abrogates disruption of hippocampal synaptic plasticity by AD-patient-derived soluble Aß in vivo. A 3H3 single-chain variable fragment (scFv) retained the binding specificity of the 3H3 IgG and, when expressed in the brains of transgenic mice using an adeno-associated virus (AAV) vector, decreased parenchymal Aß amyloid deposition in TgCRND8 mice and ADan (Danish Amyloid) cerebral amyloid angiopathy in the mouse model of FDD. These data indicate that naturally occurring human IgGs can recognize a conformational, amyloid-specific epitope and have potent anti-amyloid activities, providing a rationale to test their potential as antibody therapeutics for diverse neurological and other amyloid diseases.

Transthyretin aggregate-specific antibodies recognize cryptic epitopes on patient-derived amyloid fibrils.

Amyloidosis involves the extracellular deposition of proteinaceous amyloid fibrils and accessory molecules in organ(s) and/or tissue(s), and is associated with a host of human diseases, including Alzheimer disease, diabetes, and heart disease. Unfortunately, the amyloidoses are currently incurable, and there is an urgent need for less invasive diagnostics. To address this, we have generated 22 monoclonal antibodies (mAbs) against aggregates formed by a blood transport protein, transthyretin (TTR), which primarily forms amyloid fibrils in a patient's heart and/or peripheral nerves. Four of the mAbs, 2T5C9, 2G9C, T1F11, and TB2H7, demonstrated diagnostic potential in enzyme-linked immunosorbent assays (ELISA) by their low to sub-nanomolar cross-reactivity with recombinant wild-type (WT) and mutant TTR aggregates and lack of binding to native TTR or amyloid fibrils formed by other peptides or proteins. Notably, in the presence of normal human sera, three of the four mAbs, 2T5C9, 2G9C, and T1F11, retained low nM binding to TTR amyloid fibrils derived from two patients with familial amyloidotic polyneuropathy (FAP). The two most promising mAbs, 2T5C9 and 2G9C, were also shown by immunohistochemistry to have low nM binding to TTR amyloid deposits in cardiac tissue sections from two FAP patients. Taken together, these findings strongly support further investigations on the diagnostic utility of TTR aggregate specific mAbs for patients with TTR amyloidoses.

A binding-site barrier affects imaging efficiency of high affinity amyloid-reactive peptide radiotracers in vivo.

Amyloid is a complex pathology associated with a growing number of diseases including Alzheimer's disease, type 2 diabetes, rheumatoid arthritis, and myeloma. The distribution and extent of amyloid deposition in body organs establishes the prognosis and can define treatment options; therefore, determining the amyloid load by using non-invasive molecular imaging is clinically important. We have identified a heparin-binding peptide designated p5 that, when radioiodinated, was capable of selectively imaging systemic visceral AA amyloidosis in a murine model of the disease. The p5 peptide was posited to bind effectively to amyloid deposits, relative to similarly charged polybasic heparin-reactive peptides, because it adopted a polar α helix secondary structure. We have now synthesized a variant, p5R, in which the 8 lysine amino acids of p5 have been replaced with arginine residues predisposing the peptide toward the α helical conformation in an effort to enhance the reactivity of the peptide with the amyloid substrate. The p5R peptide had higher affinity for amyloid and visualized AA amyloid in mice by using SPECT/CT imaging; however, the microdistribution, as evidenced in micro-autoradiographs, was dramatically altered relative to the p5 peptide due to its increased affinity and a resultant "binding site barrier" effect. These data suggest that radioiodinated peptide p5R may be optimal for the in vivo detection of discreet, perivascular amyloid, as found in the brain and pancreatic vasculature, by using molecular imaging techniques; however, peptide p5, due to its increased penetration, may yield more quantitative imaging of expansive tissue amyloid deposits.

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.

AL amyloid imaging and therapy with a monoclonal antibody to a cryptic epitope on amyloid fibrils.

The monoclonal antibody 2A4 binds an epitope derived from a cleavage site of serum amyloid protein A (sAA) containing a -Glu-Asp- amino acid pairing. In addition to its reactivity with sAA amyloid deposits, the antibody was also found to bind amyloid fibrils composed of immunoglobulin light chains. The antibody binds to synthetic fibrils and human light chain (AL) amyloid extracts with high affinity even in the presence of soluble light chain proteins. Immunohistochemistry with biotinylated 2A4 demonstrated positive reaction with ALκ and ALλ human amyloid deposits in various organs. Surface plasmon resonance analyses using synthetic AL fibrils as a substrate revealed that 2A4 bound with a K(D) of ∼10 nM. Binding was inhibited in the presence of the -Glu-Asp- containing immunogen peptide. Radiolabeled 2A4 specifically localized with human AL amyloid extracts implanted in mice (amyloidomas) as evidenced by single photon emission (SPECT) imaging. Furthermore, co-localization of the radiolabeled mAb with amyloid was shown in biodistribution and micro-autoradiography studies. Treatment with 2A4 expedited regression of ALκ amyloidomas in mice, likely mediated by the action of macrophages and neutrophils, relative to animals that received a control antibody. These data indicate that the 2A4 mAb might be of interest for potential imaging and immunotherapy in patients with AL amyloidosis.

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.

Generation and characterization of anti-AA amyloid-specific monoclonal antibodies.

AA amyloidosis results from the pathologic deposition in the kidneys and other organs of fibrils composed of N-terminal fragments of serum amyloid A protein (SAA). Given that there are only limited means to visualize these deposits, we have developed a series of mAbs, 2A4, 7D8, and 8G9, that bind specifically with nanomolar affinity to a carboxy-terminal epitope generated following proteolysis of SAA that yields the predominant component of AA amyloid deposits. Notably, these antibodies do not recognize native SAA, they retain their immunoreactivity when radiolabeled with I-125 and, after injection into AA amyloidotic mice, localize, as evidenced by autoradiography and micro-single photon emission computed tomography imaging, to histologically confirmed areas of amyloid deposition; namely, spleen, liver, and pancreas. The results of our in vitro and in vivo studies demonstrate the AA fibril-selectivity of mAbs 2A4, 7D8, and 8G9 and warrant further investigation into their role as novel diagnostic agents for patients with AA amyloidosis.

Splenic plasma cells can serve as a source of amyloidogenic light chains.

Bone marrow-derived clonal plasma cells, as found in systemic amyloidogenic light chain-associated (AL) amyloidosis, are presumed to be the source of light chains that deposit as fibrils in tissues throughout the body. Paradoxically, people with this disorder, in contrast to multiple myeloma, often have a low percentage of such cells, and it is unknown whether this relatively sparse number can synthesize enough amyloidogenic precursor to form the extensive pathology that occurs. To investigate whether another hematopoietic organ, the spleen, also contains monoclonal light chain-producing plasma cells, we have immunostained such tissue from 26 AL patients with the use of antiplasma cell, antifree kappa and lambda, and anti-V(L) subgroup-specific monoclonal antibodies (mAbs). In 12 cases, there was statistically significant evidence of a monoclonal population bearing the same kappa or lambda isotype as that within the bone marrow and identical to the amyloid. Our studies have shown that the spleen may be another source of amyloidogenic light chains.