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Human Immunology and Cancer Program, Department of Medicine, University of Tennessee Center for Health Science, College of Medicine, Knoxville, TN 37920
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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1–40 peptide, but notably, do not react with these molecules in their native nonfibrillar forms. After isolation of the Abs from IGIV via fibril-conjugated affinity column chromatography, the EC50-binding value for light chains and amyloid 1–40 peptide fibrils was 
15 nM–a magnitude 200 and 70 times less than that of the unbound fraction and unfractionated product, respectively. Comparable reactivity was found in the case of those formed from serum amyloid A, transthyretin, and islet amyloid polypeptide. The purified Abs immunostained human amyloid tissue deposits and could inhibit fibrillogenesis, as shown in fibril formation and extension assays. Most importantly, in vivo reactivity was evidenced in a murine model when the enriched Abs were used to image amyloid, as well as expedite its removal. These promising experimental results suggest that fibril affinity-purified IGIV has potential as a diagnostic and therapeutic agent for patients with amyloid-associated disease. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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To date, over 20 different amyloidogenic proteins have been identified (4), but irrespective of their varied amino acid sequences, sources of origin, or biologic functions, all types of fibrils have virtually identical tinctorial and ultrastructural features; i.e., after treatment with Congo red and examination by polarizing microscopy, they exhibit a characteristic green birefringence (5), and their interaction with thioflavin T (ThT)3 results in a >100 nm red shift in this compound’s excitation spectrum (6). When negatively stained with uranyl acetate and viewed by electron microscopy, the fibrillar components are 10 nm in diameter, of indeterminate length, and consist of 2–5, often twisted, filaments arranged in parallel, with surface banding patterns indicative of a helical structure. All have a cross--pleated configuration (7, 8, 9), a property that accounts for the typical birefringent and morphologic features. Additionally, fibrils, regardless of protein composition, share conformational epitopes (10, 11, 12, 13), thus further indicating their structural commonality.
We now have found that human sera, as well as immune globulin products derived from large pools of plasma from normal donors, contain IgG Abs that also recognize a conformational epitope(s) expressed on light chains (LC), serum amyloid A (SAA), transthyretin (TTR), islet amyloid polypeptide (IAPP), and amyloid 1–40 peptide (A) fibrils, but do not when these components are in their native nonfibrillar states. We have used affinity chromatography to isolate the reactive species and report the results of our studies that have demonstrated the capability of these Abs to inhibit fibrillogenesis, as well as immunostain amyloid deposits. Moreover, we provide evidence that these molecules have in vivo activity in that they can be used to image amyloid and accelerate its destruction when given to mice bearing human amyloidomas. Our findings suggest that fibril-reactive IgG Abs, due to their unique specificity and human origin, could have novel diagnostic and therapeutic potential for patients with amyloid-associated disease.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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Human immune globulin preparations (Gammagard S/D, Polygam S/D, and Panglobulin) were products of Baxter Healthcare. The murine IgG
protein MOPC 31-C was purchased from Sigma-Aldrich. Recombinant 
6 LC variable region (rV6) components Jto and Wil were produced in an Escherichia coli expression system (14), and recombinant mutant (V30M) TTR was provided by Dr. J. N. Buxbaum Scripps Research Institute, La Jolla, CA. The A peptide, its Cys-1 analog, and IAPP were synthesized at the Keck Biotechnology Center (Yale University, New Haven, CT).
Amyloid fibrils used for amyloidoma formation in mice were extracted from tissue samples obtained postmortem from patients with AL-, AL-, AA-, or ATTR (T60A mutation)-associated amyloidosis, and their chemical nature was determined by automated amino acid sequencing and tandem mass spectrometry (15).
Soluble rV6 molecules were biotinylated with a 12-fold molar excess of a 10 mM stock solution of EZ-Link Sulfo-NHS-Biotin (Pierce), and, after incubation at room temperature for 45 min, the unreacted SHB was removed by dialysis. The biotinylated A Cys-1 analog was provided by Dr. R. Wetzel (University of Tennessee Graduate School of Medicine, Knoxville, TN).
LC were sterile-filtered using a 0.22-µm polyvinylidene fluoride 25-mm Millex-GV syringe-driven filter unit (Millipore) and found by Sephadex G25 (Amersham Biosciences) gel filtration to consist of monomers and dimers; no higher-order aggregates were present (14). The IAPP and A preparations were disaggregated by sequential exposure to trifluoroacetic acid and hexafluoroisopropanol (16). Before study, all proteins or peptides were centrifuged at 20,800 x g for 25 min, and, in the case of A, 50,000 x g for 18 h.
Fibril preparation
In vitro-generated LC, IAPP, and A fibrils were prepared as described previously (13). TTR fibrils were formed within 7 days under similar conditions from solutions of soluble protein (2 mg/ml) incubated at 37°C in a 0.05 M sodium acetate/0.1 M KCl buffer (pH 4.4), plus 0.001 M EDTA (maximum ThT signal was obtained at this time point). All fibril samples were harvested by centrifugation, sonicated (2 x 30 s bursts) with a probe sonic disrupter (Teledyne/Tekmar), aliquoted, and stored at 4°C for up to 2 wk or long-term at –20°C. These synthetic fibrils were used in all experiments except the amyloidoma studies.
Preparation of fibril affinity column
Ten milliliters of LC (rV6 Jto) sonicated fibrils in PBS (3 mg/ml) were conjugated at room temperature for 3 h to a 10-ml packed bed volume of N-hydroxysuccinimide-activated Sepharose 4 fast-flow agarose matrix (Amersham Biosciences), and the column was equilibrated with PBS.
Isolation of fibril-reactive IgG Abs
Lyophilized immune globulin (5 g) was reconstituted in 100 ml of sterile water for injection (USP). The sample was passed through a 0.22-µm filter, diluted 5-fold with PBS to yield a final concentration of 10 mg/ml, and loaded onto the fibril-conjugated column. Weakly binding or unbound (residual) protein was collected, and then the column was washed with PBS, and the fibril-bound (enriched) Abs were eluted in 1-ml portions using 0.1 M glycine buffer (pH 2.7); the fractions were neutralized by addition of 1 M Tris-HCl (pH 9). The concentration of IgG in each sample was determined based on OD at 280 nm, using an E1%280 of 1.30 and a Mr of 150,000 kDa. Samples containing the enriched Abs were pooled and concentrated to 1 mg/ml with a PL-30 Centricon (Millipore) apparatus.
The IgG subclass composition of the affinity-purified Abs was determined using the Human IgG Subclass Profile ELISA Kit (Zymed Laboratories).
Europium-linked immunosorbent assay (EuLISA)
The dissociation-enhanced lanthanide fluoroimmunoassay (17) incorporating europium (Eu3+)-streptavidin and time-resolved fluorometry (DELFIA System; PerkinElmer Life Sciences) was essentially as described in Ref. 13 , except for the reduced concentration of BSA (1%) used for blocking and in the assay buffer. For the binding studies, sera or IgG fractions were serially diluted in activated, high-binding microtiter plate wells (Costar) coated with 400–500 ng of soluble or fibrillar protein or peptide, respectively. For competition studies, the concentration of Ab remained constant (80 nM), and inhibitors were serially diluted (1.2–0.0002 mg/ml). A biotinylated goat anti-human IgG reagent (
-chain specific; Sigma-Aldrich) served as secondary Ab, and, after addition of a Eu3+-streptavidin conjugate followed by the releasing enhancement solution, Eu3+ time-resolved fluorescence was measured with a PerkinElmer Victor2 1420 Multilabel Counter. The amount (fM) of lanthanide released was calculated from a standard curve using known concentrations of Eu3+. All measurements in this and other assays were done in triplicate (error bars in the figures represent SD).
Fibrillogenesis assay
ThT fluorescence intensity was monitored to determine whether IgG preparations could block conversion of soluble proteins or peptides into amyloid fibrils. For studies involving LC (rV6 Wil) or IAPP, wells of an ultra low-binding plate (Costar) were filled first with 5 or 50 µM, respectively, of the native molecules and then with serially diluted (0.5 µM–250 nM) enriched, residual, or unfractionated Ab in PBS containing 0.05% sodium azide (PBSA) plus 30 µM ThT (control wells contained IgG preparations or precursor protein/peptide). The plates were then sealed and incubated at 37°C. Fluorescence was measured daily with a FL600 microplate reader (Bio-Tek Instruments). In the case of A, 90 µM soluble peptide and 1 µM enriched, residual, or unfractionated IgG were added to Eppendorf tubes, and, at designated time intervals, aliquots were removed and placed in microtiter plate wells, followed by addition of ThT.
Fibril extension assays
Wells of activated high-binding microtiter plates each were filled with 400 ng of sonicated LC, A, or IAPP fibrils in PBSA and dried as described previously (16). The wells were blocked by addition of 1% gelatin in PBSA for 1 h at 37°C and then filled with 50 µl of serially diluted IgG (4 µM–31 nM) in wash buffer (PBS plus 0.02% Tween 20). Immediately afterward, 50 µl of soluble biotinylated LC protein (250 nM), A peptide (50 nM), or unbiotinylated IAPP (28 µM) was added, and the plate was incubated for 3 h. For experiments with LC and A, the wells were filled with the Eu3+-streptavidin conjugate, then with enhancement solution, and free Eu3+ was quantitated by time-resolved fluorometry. In the case of IAPP, ThT was added to reaction wells and its fluorescence monitored.
Immunohistochemistry
Areas of amyloid deposition in autopsy-derived human tissues were identified by Congo red staining and microscopic evaluation (5). Immunohistochemical analyses were performed with the Elite ABC kit (Vector Laboratories). Deparaffinized serial 4-µm-thick sections, mounted on poly-L-lysine-coated slides, were subjected to Ag retrieval (30 min in boiling distilled water) and then endogenous peroxidase activity blocked with 0.3% H2O2. After washing with PBS, the tissues were exposed to fibril-reactive Ab (0.1 mg/ml) that had been premixed with an appropriate dilution of goat anti-human IgG biotinylated conjugate (for control purposes, the antiserum also was absorbed with a 10-fold excess of sonicated LC fibrils), and the slides were incubated for 48 h at room temperature. Sections were washed, subjected first to the avidin DH HRP H complex, and then to the peroxidase substrate solution, followed by a hematoxylin counterstain.
For studies involving cerebral cortical tissue from a patient with Alzheimer’s disease, amyloid deposits in 6-µm-thick sections were identified after Congo red staining and confirmed to contain A-related protein immunohistochemically (after pretreatment for 5 min at room temperature with 95% formic acid) using a murine anti-A mAb (NCL-B-amyloid; Novacostra Laboratories) in conjunction with the Vector Immuno PRESS detection system (Vector Laboratories). Ag retrieval involved heating the specimen at 90°C for 20 s in Dulbecco’s saline solution (pH 7.4) using a Digital Decloaking Chamber (Biocare Medical), as per the manufacturer’s instructions, followed by exposure first to 4% SDS for 1 h at 37°C and then to a 0.25% solution of pepsin (Pepsin Kits; BioGenex) for 30 min at 37°C. For immunostaining, sections were incubated for 18 h at room temperature with the enriched immune globulin i.v. (IGIV) preparation (0.02 mg/ml) that had been premixed with an appropriate dilution of a goat anti-human IgG-biotinylated conjugate (Vector Laboratories). After washing with PBS, the murine anti-A mAb-treated tissue was overlaid with an HRP-conjugated secondary Ab followed by addition of the substrate-chromagen solution. The IGIV-treated section was subjected first to the Avidin DH-HRP-H complex, then to the peroxidase substrate (Elite ABC kit; Vector Laboratories), and finally, as a counterstain, to hematoxylin.
Amyloidoma formation in mice
Eight week-old BALB/c or SCID mice were injected s.c. between the scapulae with 50–100 mg of human AL or ATTR extracts, as described previously (12). The animals were treated in accordance with National Institutes of Health regulations under the aegis of a protocol approved by the University of Tennessee’s Animal Care and Use Committee.
Radioimaging studies
Fibril affinity-purified (enriched) IgG Abs were labeled with I-125 (1 mCi (37 MBq) per milligram of protein) by the chloramine T method; residual isotope and protein aggregates were removed by size-exclusion liquid chromatography. Imaging data were recorded using a micro single photon emission computed tomography (SPECT) instrument capable of high-level spatial resolution to 1.7 mm (18). For each study, 60 projections were acquired at 6° intervals, and the images were reconstructed using an implementation of the EM-ML algorithm (19). After completion of the SPECT study, animals were placed in the high-resolution (to 50 µm) micro-computed tomography (CT) apparatus, and 180 projections were collected. Reconstructed SPECT and CT images were coregistered using I-125-filled capillaries as fiducials.
To determine the biodistribution of the labeled reagent, samples of skin, muscle, abdominal fat, liver, pancreas, kidney, spleen, heart, and lung, as well as the amyloidoma, were harvested, placed into tared vials, and the radioactivity was measured. The primary index values were expressed as percentage of injected dose per gram of tissue.
Statistical analyses
Data were compared using a one-way ANOVA with confidence limits of 95%.
Assurances
Studies involving human specimens were in accordance with a protocol approved by the University of Tennessee Medical Center’s Institutional Review Board.
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Results |
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We initially had used an ELISA-based method to demonstrate that AL amyloidoma-bearing mice developed IgG Abs that recognized homologous, as well as other LC-related fibrils (12). Furthermore, with this technique, we found that sera from some normal humans also contained such components. In an effort to determine whether these Abs bound to fibrils formed from amyloidogenic precursor proteins other than LCs, we used another detection system that would provide increased sensitivity and dynamic range; namely, time resolved fluorometry (17) that uses the chelated lanthanide label europium (Eu3+). As detailed in Materials and Methods, this sandwich-type immunosorbent assay (EuLISA) involves the capture of reactive Abs onto fibril-coated wells and their detection by sequential addition of a biotinylated anti-human IgG reagent, a streptavidin Eu3+ complex, and an Eu3+-releasing agent. Finally, the amount of fibril-bound Ab is determined based on the magnitude of Eu3+ fluorescence.
Analyses by EuLISA of serum specimens obtained from 10 healthy adults revealed varying titers of IgG Abs that recognized LC fibrils. Additionally, the five most reactive samples also bound those formed from SAA, TTR, IAPP, or A. The results obtained in two representative cases are illustrated in Fig. 1. On this basis, we assayed Cohn Fraction II -globulin and three different pharmacologic sources of immune globulin (IGIV) and found that fibril-binding Abs were present in all.
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To isolate the active species from IGIV, we used affinity chromatography where LC fibrils were conjugated to Sepharose beads. Passage of the reconstituted, filtered solution through the column yielded two fractions: the first consisted of unbound protein present in the PBS filtrate; and the second, the fibril-bound Ab eluted by the acidic buffer (designated residual and enriched IgG, respectively). The distribution of the IgG 1, 2, 3, and 4 subclasses in both pools was comparable and similar to that found in normal serum. Based on the protein concentrations of the filtrate and eluate, the isolated Abs represented 0.2% of the IgG molecules in the IGIV product.
The affinity of the purified Abs for LC, AA, TTR, IAPP, and A fibrils was considerably greater than was that of the unfractionated preparation, thus indicating that virtually all of the fibril-reactive IgG was isolated by the chromatographic procedure (Fig. 1). The EC50-binding value of the affinity-purified preparation for LC and A fibrils (15 nM) was 70 and 200 times less than that of the unfractionated (1 µM) and residual (3 µM) materials, respectively. For AA, the EC50 of the enriched fraction was 23 times less (46 nM) than that of the unfractionated (1 µM), whereas for TTR and IAPP, the EC50 was not obtained; however, similar binding was noted. In other experiments, IgG Abs eluted from an A fibril-conjugated column reacted with comparable EC50 values to A and LC fibrils as did those derived from the LC affinity matrix.
As shown in Fig. 2, A and B, affinity-purified IgG reacted specifically with LC and A fibrils in a competition assay, but remarkably, did not recognize their soluble counterparts (even when tested at 125 molar excess). Furthermore, LC, TTR, and IAPP fibrillar components were efficient inhibitors of the interaction with A fibrils (Fig. 2C), whereas human sera or other soluble proteins (thyroglobulin, collagen, insulin), as well as nonamyloidogenic-aggregated molecules (dsDNA, reduced and alkylated OVA, elastin), at concentrations of up to 1.5 mg/ml, had no effect.
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250 nM, whereas the unfractionated portion had no effect (Fig. 3A). This was not a transient or kinetic response, because the inhibition noted at 18 h (the time at which maximum fibril formation occurred in the absence of Ab) persisted over at least 2 days. Similarly, these molecules blocked A fibril formation (Fig. 3B). As for IAPP, based on kinetic traces, the enriched IgG Abs slowed fibrillogenesis by halting conversion of the less structured intermediate form (16) into fibrils, as seen by the single and biphasic nature of the curves generated in the presence or absence of Ab, respectively (Fig. 3C). Additionally, in contrast to residual or unfractionated IgG, the isolated preparation inhibited IAPP fibrillogenesis in a dose-dependent manner with an IC50 value of 0.25 µM (Fig. 3D). Control experiments with preformed LC, A, and IAPP fibrils indicated that the fluorescence emission from bound ThT was not quenched nonspecifically by the Ab.
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were coincubated with the enriched fraction for 14 days, dried onto grids, and stained with uranyl acetate. In the case of LC and A, the affinity-purified IgG Abs totally blocked fibril formation (data not illustrated). For IAPP, electron micrographs revealed less ordered, dense aggregates characteristic of the assembly intermediate (16), whereas, in the presence of unfractionated Ab or PBS, these preparations contained ultrastructurally typical fibrillar material (Fig. 3E). Furthermore, purified IgG limited growth of LC, A, and IAPP fibrils in a dose-dependent fashion, with IC50 values in the extension assays of 1.6 µM, 0.05 µM, and 0.9 µM, respectively (Fig. 3, F–H).
The purified IgG Abs also recognized amyloid in tissue, as documented immunohistochemically. These molecules immunostained the green birefringent congophilic deposits present in the renal glomeruli, pericardium, ovary, myocardium, and pancreas from patients with AL, AL, AA, ATTR, and AIAPP amyloidosis, respectively, as well as A-containing cerebral cortical plaques in brain tissue obtained from an individual with Alzheimer’s disease. This reactivity was totally abolished when the enriched Abs were absorbed with LC fibrils (Fig. 4). Furthermore, there was little or no staining with the residual fraction.
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To determine whether the fibril-reactive Abs would have in vivo activity, radiolabeled affinity-purified IgG was used in imaging studies involving the murine amyloidoma model (12). Sets of three BALB/c mice were given 15-µg (160 µCi, 6 MBq) injections into the tail vein of 125I-labeled-enriched IgG 1 wk after induction of amyloidomas containing 50 mg of human AL or ATTR extracts, and mice were euthanized 72 h later (nonamyloidoma-bearing animals served as controls) and scanned by high-resolution microSPECT (18). Localization of the radioiodinated Abs to the amyloid was apparent in coregistered microSPECT/microCT images that revealed the radioisotope to be concentrated within the AL (Fig. 5) and ATTR amyloidomas (not illustrated), with only minimal background activity occurring in other organs or tissues. The preferential binding of the radioiodinated IgG Abs with the amyloid also was indicated by the biodistribution of the labeled reagent, where the specific activities of the harvested tumors was 24% of the injected dose per gram of tissue, a value at least two times greater than found at any other site and significantly more than that of skin, muscle, and fat (p < 0.05) (Table I). In contrast, the uptake of an irrelevant radiolabeled IgG monoclonal protein (MOPC 31-C) was <1%. The presence of radioiodinated Ab in other areas was comparable to that of a nonamyloidoma-bearing mouse.
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Pairs of SCID mice bearing 100-mg, s.c. amyloidomas composed of AL, AL, or ATTR extracts were given, on days 0, 2, 4, 6, 8, and 10, a series of 0.25-ml s.c. injections of unfractionated or enriched IGIV (1 mg/ml) in each flank (another group received an equivalent amount of PBS alone). Eighteen days after the last injection, the AL, AL, and ATTR amyloidomas in animals that received the unfractionated IGIV were reduced in size 35, 64, and 30%, respectively, compared with those that were given PBS alone. This effect was more pronounced (63, 99, and 65%) with the enriched IGIV preparation (Fig. 6). The mean of tumor weights in the enriched, unfractionated, and PBS control mice was 0.14 ± 0.09, 0.22 ± 0.07, and 0.40 ± 0.17, respectively, and a pairwise comparison of the extent of amyloidolysis revealed a significant difference between the enriched and control groups (p < 0.05). As noted in studies with the amyloid-reactive 11–1F4 mAb (12), pronounced infiltration of activated neutrophils, mononuclear cells, and macrophages was seen in the resolving amyloidomas of the IgG-treated animals. Microscopic examination of Congo red-stained tissues showed that green birefringent material in both groups was confined to the amyloidomas and was not found in any other site.
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Discussion |
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-sheets that lead to protofilaments/protofibrils; and, finally, interaction of the components to form the mature fibril (7, 8, 9). The profound structural changes that occur as normally folded globular proteins make this transition are manifested by differential epitope expression (10, 11); e.g., one class of Ab may recognize sequence-specific linear epitopes exposed on the fibril, as well as the partially unfolded amyloidogenic intermediate and native precursor protein; a different type reacts with neoepitopes present on the fibril and assembly intermediates (i.e., antigenic regions buried in the native molecule that become exposed as a result of protein unfolding); and yet another binds to a conformational epitope present on all fibrils, irrespective of primary structure.
mAbs representative of groups 1, 2, and 3 have been generated against A, TTR, and LC fibrils and are exemplified by reagents that recognize, respectively, a site within the exposed N-terminal six residues of the native and fibrillar forms of A (20), a neoepitope on TTR (21), and a common fibril-specific epitope (12, 13). Notably, the polyclonal fibril-reactive IgG Abs used in our studies contain molecules representative of group 3 that specifically recognize a generic epitope expressed by fibrils formed from at least five different amyloidogenic precursors (LC, SAA, TTR, IAPP, and A), but do not react with these proteins or peptides in their native states. In this respect, they differ from, for example, the anti-A-reactive components found in human sera (22, 23, 24), an immune globulin product (25, 26, 27), the IgM mAbs generated from peripheral blood lymphocytes (28), and other types of anti-A reagents (29). Although other investigators have identified Abs that differentiate between the soluble and fibrillar configurations of A (30, 31), it remains to be established whether these reagents bind heterologous fibrils as does IGIV.
The amyloid-reactive IgGs that we have identified in human sera are unlike other polyreactive Abs (32) in that they are fibril-specific; furthermore, they do not bind nonamyloid-aggregated or fibrillar macromolecules. These components seemingly represent yet another example of autoreactive molecules formed as part of a humoral immune response to an endogenous or exogenous antigenic stimulus. One intrinsic source of fibrils may be those formed from normally soluble proteins that have the propensity to self-associate and become fibrillogenic as part of the aging process (3). Alternatively, extrinsic fibrillar materials (33, 34, 35, 36, 37, 38, 39, 40) may themselves be immunogenic or, because fibrillogenesis is a nucleation-dependent process (41), serve as seeds to generate fibrils from amyloidogenic precursor proteins.
The functional significance of IgG-associated pan fibril-reactive Abs is presently unknown. As shown in fibril formation and extension assays, these molecules can act as inhibitors of fibrillogenesis. Furthermore, such Abs may facilitate destruction of amyloid deposits or clear fibrillar aggregates from the circulation, analogous to the removal of misfolded intracellular proteins by molecular chaperones.
As yet, there are only limited methods (42, 43) to document radiographically the extent or presence of amyloid deposition (or its resolution); thus, there is a critical need for an objective means to ascertain a patient’s response to treatment and/or to determine whether relapse has occurred. The use of radiolabeled fibril-reactive Abs as imaging agents appears promising, as evidenced by the selective uptake of radioiodinated fibril affinity-purified IgG by human AL and ATTR amyloidomas. Furthermore, the distribution data, coregistered SPECT/CT images, and the favorable signal-to-noise ratio have indicated the diagnostic potential of this reagent for patients with systemic forms of amyloidosis. For those with Alzheimer’s disease, fibril-reactive, single chain Fv components may prove more suitable because their lower m.w. would facilitate passage into the brain.
Currently, there are few therapeutic options available for patients with amyloid-associated disease. Thus, the use of fibril-reactive IgG Abs of human origin to facilitate removal of amyloid deposits may prove to be an effective means of treatment. In this regard, we have shown that the low titers of fibril-reactive Abs found in the sera of patients with AL or ATTR amyloidosis could be increased significantly when enriched IgG was added to the specimens. This enhanced reactivity was evidenced, not only against LC or TTR fibrils, but also to protein extracted from the patient’s own amyloid-laden tissue. Based on our experimental results, we posit that amyloid resolution resulted from an in situ three-step process that included the following: 1) the binding or opsonization of fibrils by the fibril-reactive IgG Abs; 2) attraction and activation of neutrophils and macrophages via Fc receptor interaction; and 3) enzymatic and/or chemical proteolysis of the amyloid by endopeptidases or reactive oxygen species, respectively.
The potential of passive immunotherapy to effect amyloidolysis also has been evidenced in transgenic murine models of Alzheimer’s disease (31, 44, 45). Because the Abs administered reacted with the A monomer (as in the case of those generated via vaccination with A-related peptides; Ref. 45), other investigators have attributed this response to the binding and/or sequestration of native A (46). Given the fact that fibril-reactive IgG does not react with this or other nonfibrillar forms of amyloidogenic precursor molecules, infusion of these Abs could be clinically advantageous because it is unlikely that potentially harmful immune complexes would be formed. However, it remains to be determined whether an amyloidolysis-associated inflammatory response would be deleterious.
In summary, we have isolated high-affinity Abs from human pooled immune globulin that specifically recognize fibrils formed from five different amyloidogenic precursors and have documented their binding specificity through the use of a sensitive fluoroimmunoassay, as well as immunohistochemically where they immunostained amyloid deposits in tissue. Furthermore, these molecules could inhibit fibrillogenesis, as shown in fibril formation and extension assays. In other studies involving an in vivo experimental murine model, the purified Abs, when radiolabeled, served as diagnostic reagents capable of imaging amyloid deposits. Moreover, therapeutic activity was evidenced by their acceleration of amyloidolysis in animals bearing human amyloidomas. Based on our experimental results, we suggest that fibril-reactive IgG Abs may provide a novel diagnostic and therapeutic modality to improve the invariably poor prognoses of patients with amyloid-associated disease.
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Acknowledgments |
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tissue samples; and Ron Wetzel for helpful discussions. |
Disclosures |
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Footnotes |
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1 This work was supported in part by U.S. Public Health Service Research Grants CA 10056 and EB00789 from the National Cancer Institute and National Institute of Biomedical Imaging and Bioengineering/National Institute of Neurological Disease and Stroke, respectively, and the Aslan Foundation. R.H. was the recipient of a Brian D. Novis Award from the International Myeloma Foundation. A.S. is an American Cancer Society Research Professor. 
2 Address correspondence and reprint requests to Dr. Alan Solomon, University of Tennessee Graduate School of Medicine, 1924 Alcoa Highway, Knoxville, TN 37920. E-mail address: asolomon{at}mc.utmck.edu
3 Abbreviations used in this paper: ThT, thioflavin T; LC, light chain; SAA, serum amyloid A; TTR, transthyretin; IAPP, islet amyloid polypeptide; A, amyloid 1–40 peptide; EuLISA, europium-linked immunosorbent assay; IGIV, human immune globulin i.v.; SPECT, single photon emission computed tomography; CT, computed tomography.
Received for publication September 19, 2005. Accepted for publication March 10, 2006.
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References |
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-sheet structures. Amyloid 2: 1-6. [Medline]6 light chains: correlation with fibrillogenicity. Biochemistry 38: 14101-14108. [Medline]-amyloid peptide. Proc. Natl. Acad. Sci. USA 93: 452-455. and Alzheimer’s disease. Ann. Neurol. 49: 808-810. [Medline]-peptide antibody in Alzheimer disease. Neurology 57: 801-805. peptide: a potential treatment for Alzheimer’s disease. Ann. Neurol. 52: 253-256. [Medline]-amyloid antibodies block -amyloid fibril formation and prevent -amyloid induced neurotoxicity. Brain 126: 1935-1939. -amyloid for the treatment of Alzheimer’s disease. J. Neurol. Neurosurg. Psychiatry 75: 1472-1474. from healthy adults. Neurobiol. Aging 26: 597-606. [Medline]-amyloid (A) can inhibit A aggregation and prevent A-induced neurotoxicity. Biochemistry 43: 6959-6967. [Medline]-amyloid by vaccination of patients with Alzheimer disease. Nat. Med. 8: 1270-1275. [Medline]-amyloid peptide for protection against Alzheimer’s disease-like neuropathology. Proc. Natl. Acad. Sci. USA 100: 2023-2028. -amyloid immunotherapy. Curr. Opin. Immunol. 16: 599-606. [Medline] antibody alters CNS and plasma A clearance and decreases brain A burden in a mouse model of Alzheimer’s disease. Proc. Natl. Acad. Sci. USA 98: 8850-8855.
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, •) or fibril affinity-purified (right) (•), unfractionated (
), and unbound (
) IgG and then to biotinylated anti-human 
) vs their fibrillar counterparts (•) to inhibit, in a dose-dependent fashion, the binding of fibril affinity-purified (enriched) IgG to LC- (A) and A
), residual (
), and unfractionated (
). C, Kinetic traces of IAPP fibril formation in the presence or absence of enriched, residual, or unfractionated IgG (1 µM) as measured by ThT fluorescence. D, Comparison of the dose-dependent inhibitory effect of enriched (
), and unfractionated (
