Edited by: Harry W. Schroeder, University of Alabama at Birmingham, USA
Reviewed by: Jayanta Chaudhuri, Memorial Sloan Kettering Cancer Center, USA; Antonio La Cava, University of California Los Angeles, USA
*Correspondence: Jonathan S. Wall, University of Tennessee Graduate School of Medicine, 1924 Alcoa Highway, Knoxville, TN 37920, USA. e-mail:
This article was submitted to Frontiers in B Cell Biology, a specialty of Frontiers in Immunology.
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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
AA amyloidosis is characterized by the pathological deposition in various body tissues, particularly the kidneys, spleen, and liver (Gillmore et al.,
From a clinical standpoint, there is a need for an objective means to document the extent of AA amyloid deposition or its resolution in order to ascertain a patient’s response to treatment and/or if relapse has occurred. In this regard, routine radiographic techniques (CT, MRI, and ultrasound) are not particularly informative or “amyloid specific”; furthermore, the deposits are rarely visualized using available nuclear medicine agents. Although European investigators have imaged AA amyloid by planar gamma scintigraphy and single photon emission computed tomography (SPECT) using 123I-labeled serum amyloid P-component (Hawkins et al.,
Another strategy involves use of specific radiolabeled fibril-reactive antibodies as imaging agents. A precedent for this approach has been established utilizing mAb 11-1F4, which recognizes an amyloid fibril-dependent, conformational epitope on immunoglobulin light chains, but is non-reactive with the natively folded molecules. This mAb, when radiolabeled with the positron-emitting isotope I-124, has been shown by PET/CT to image AL amyloid, first in an animal model (Wall et al.,
Given the necessity to monitor the presence and biodistribution of AA amyloid in the major target organs of patients with AA amyloidosis, we have developed a series of mAbs, designated 2A4, 7D8, and 8G9, that bind specifically to AA fibrils, but not the normal circulating precursor protein, and have defined the structural basis for their specificity. Notably, these reagents, when radiolabeled with I-125, visualized fibrillar deposits in a transgenic murine model of AA amyloidosis (Solomon et al.,
A peptide containing amino acids 71–75 of murine SAA, with two artificial N-terminal amino acids added for ease of coupling (
AJ mice (Jackson Labs) were immunized with 50 μg of the peptide conjugate in complete Freund’s adjuvant, followed by boosts 14 and 28 days later with another 50 μg in incomplete Freund’s adjuvant. Animals were bled on day 35, where the anti-peptide titer, as determined by ELISA, was >1/10000. Splenocytes from one such mouse were fused to SP2/0 cells and the resulting hybridomas screened by ELISA for reactivity to the CGGHEDT component, as well as for lack of reactivity to a peptide that included an additional five downstream residues of the murine SAA sequence (GHEDTMADQE). Three hybridomas, 2A4, 7D8, and 8G9, were identified, stabilized, and cloned by three rounds of limiting dilution. Antibodies were generated in ascites and following delipidation were isolated from a 50% (w/v) ammonium sulfate precipitate by centrifugation. The antibody precipitate was dialyzed against PBS and then purified on a Protein G column.
Purified antibodies were diluted to 1 mg/ml in PBS and biotinylated using EZ-Link NHS-PEG4-Biotin, No Weigh Format (ThermoFisher Scientific), according to the manufacturer’s specifications, except that the ratio of biotin to antibody was reduced to a 10-fold molar excess.
Murine and human AA amyloid extracts were isolated from amyloidotic tissues by a serial flotation procedure, as described previously (Pras et al.,
All animal experiments were performed in accordance with Institutional Animal Care and Use Committee-approved protocols.
Six micrometer-thick sections, cut from formalin-fixed, paraffin-embedded tissues, were subjected to antigen retrieval using the High pH Target Retrieval™ system (Dako). After a 30-min incubation at 90°C, followed by 20 min at room temperature, the tissues were immunostained overnight at 4°C with a 3-μg/ml solution of biotinylated anti-AA amyloid-specific mAb in 0.05% Tween/PBS. The reaction was developed using the Vectastain ABC kit (Vector Labs). The presence of amyloid was confirmed by staining with a 0.1% alkaline Congo red solution, where the characteristic green birefringence was seen upon examination using a Leica DM500 light microscope fitted with cross-polarizing filters. Digital microscopic images were acquired with a cooled CCD camera (SPOT, Diagnostic Instruments).
Wells of NUNC Maxisorb plates (eBioscience) were coated with extracts of murine AA amyloid fibrils (8 μg/ml) or the two synthetic peptides (3 μg/ml) and incubated overnight at 37°C. The dried wells were washed ×1 with assay buffer (PBS containing 0.05% Tween 20) and blocked with 200 μl of 1% BSA in PBS at 37°C for 1 h, followed by addition of the anti-AA amyloid-specific and control mAbs titrated in triplicate over the range of 1 M × 10−11 M–1 M × 10−6 M (to determine background values, wells were filled only with antibody). After a 1-h incubation at 37°C, the wells were washed ×2, filled with 100 μl of a 1:5000 dilution of goat anti-mouse HRP conjugate and, following another 1-h incubation at 37°C and washes, 100 μl of ABTS in 0.06% H2O2 were added. The absorbance at 405 nm was measured 30 min later with a Wallac Victor3 plate reader (PerkinElmer) and the data analyzed using SigmaPlot (SPSS Inc.).
Quadruplicate rows of NUNC Maxisorb wells were coated with 50 μl of murine or human renal or splenic AA fibrils suspended in PBS (8 μg/ml), the plates dried overnight at 37°C, and the wells blocked for 1 h at 37°C with 1% BSA/PBS (200 μl/well). The AA samples used for competition were suspended in assay buffer containing 1% BSA to make a 2× stock solution (800 μg/ml) and then serially diluted to a final concentration of 1 × 10−11 M. Anti-AA amyloid-specific and control mAbs were diluted with assay buffer to form a 2× stock solution and added to the human and murine AA fibril preparations to yield a final concentration of 50 nM and a starting 400 μg titer of competitor. Next, the competitor-mAb solutions were transferred to the AA fibril-coated plates and left for 1 h at 37°C [to establish maximum binding, one row of wells contained mAb alone (no competitor) and non-specific (background) binding was measured in those with antibody, but no fibrils]. After two washes, 100 μl of a 1:5000 dilution of goat anti-mouse HRP-conjugated secondary antibody was added and the plates incubated for 1 h at 37°C, washed, and the reaction developed by addition of 100 μl of ABTS in 0.06% H2O2. After 30 min, the absorbance was measured at 405 nm with the microplate reader. Data were analyzed using Excel and SigmaPlot, with each point representing the mean of triplicate values corrected for non-specific binding.
Forty nanomolar of anti-AA and control mAbs were labeled with 2 mCi of reductant-free 125I (Perkin Elmer), using limiting amounts of chloramine T (Wall et al.,
The biodistribution of the radiolabeled mAbs was determined in groups composed of three control or amyloidotic mice. Each animal was injected with 6 μg of 125I-labeled mAb (∼150 μCi) in the lateral tail vein and, after 48 or 72 h, given a 200-μl i.v. dose of Fenestra VC™ (Advanced Research Technologies). Thirty minutes later, the mice were euthanized by isoflurane overdose and SPECT/CT images acquired.
Single photon emission computed tomography data were collected with a microCAT II + SPECT dual modality imaging apparatus equipped with a 1-mm-pore diameter pinhole collimator (Siemens Preclinical Solutions). For imaging, the two detectors (composed of a 50-mm-diameter Hamamatsu R2486-02 multi-anode photo-multiplier tube coupled to a 1-mm × 1-mm × 8-mm CsI (Tl) crystal array arranged on a 1.2-mm2 grid) were positioned ∼45 mm from the center of rotation. Each SPECT dataset comprised 45 projections collected over 360° during the course of ∼50 min. Images were reconstructed using an implementation of the expectation maximization–maximum likelihood (EM–ML) algorithm.
After acquisition of SPECT data, high-resolution CT images were obtained with the microCAT II scanner that had a circular orbit cone beam geometry and was equipped with a 20- to 80-kVp microfocus X-ray source that captured a 90-mm × 60-mm field of view using a 2048 × 3072 CCD array detector. Each CT dataset, composed of 360 projections at 1° azimuths, was procured over 8 min. Images were reconstructed in real-time on isotropic 77-μm voxels using an implementation of the Feldkamp backprojection algorithm. To facilitate co-registration of the reconstructed SPECT and CT images, Co-57-sealed sources were placed on the imaging bed and datasets were visualized and co-registered manually with a 3-D image analysis software package (Amira, Version 3.1: Mercury Computer Systems).
Tissue samples harvested from AA amyloid-bearing and control mice injected with the 125I-labeled mAbs were placed into tared vials, weighed, and the radioactivity measured. The primary index values were expressed as % injected dose/g tissue (% ID/g).
Six micrometer-thick sections cut from formalin-fixed, paraffin-embedded blocks of tissue obtained from mice sacrificed 48 h post-injection of 125I-labeled mAb were placed on Probond microscopic slides (ThermoFisher), dipped in NTB-2 emulsion (Eastman Kodak), stored in the dark, and developed after a 96-h exposure. The sections were counterstained with hematoxylin and eosin, cover-slipped with Permount (ThermoFisher), and examined by light microscopy. In addition, consecutively cut sections were stained with Congo red and viewed under cross-polarized illumination or immunostained using the AA-reactive mAbs as primary reagents. Digital photographs were taken and evaluated with the Image Pro Plus software package (MediaCybernetics).
The primary structures of mouse, human, and three other mammalian AA proteins are provided in Figure
mAb | EC50 values | |||
---|---|---|---|---|
Amyloid extracts | Synthetic peptides | |||
Mouse AA |
Human AA |
HEDT |
HEDTMADQ |
|
2A4 | 4.09 | 26.4 | 3.4 | >>100 |
7D8 | 1.84 | 13.3 | 2.3 | >>100 |
8G9 | 5.64 | 31.7 | 4.0 | >>100 |
To ensure that the interaction of the mAbs with AA fibrils did not result from an “artificial” neo-epitope formed as a consequence of adsorbing the material onto the microplate well, competitive binding studies were performed using soluble mouse and human AA extracts (Figures
Although the mAbs were generated using the murine SAA GHEDT peptide as the immunogen, all showed high avidity for mouse and human AA amyloid with slightly higher affinity for the murine (HEDT) versus human (AEDS) sequences. To better understand the nature of the antibody binding sites, we performed studies using alanine (Ala)-substituted peptides as substrates in the ELISA (Figure
The
The capability of mAbs 2A4, 7D8, and 8G9 to bind AA amyloid
The specific localization of the 125I-labeled mAbs within amyloid was evidenced autoradiographically, where dense black deposits were seen around hepatic vasculature and sinusoids, splenic follicles, and renal papilla (Figure
2A4 | 7D8 | 8G9 | MOPC 141 | |||||
---|---|---|---|---|---|---|---|---|
Mean % ID/g | SD | Mean % ID/g | SD | Mean % ID/g | SD | Mean % ID/g | SD | |
Skin | 0.29 | 0.07 | 0.20 | 0.06 | 0.16 | 0.09 | 0.07 | 0.04 |
Muscle | 0.24 | 0.08 | 0.18 | 0.06 | 0.16 | 0.08 | 0.05 | 0.04 |
Liver | 30.77 | 5.96 | 22.66 | 4.38 | 22.87 | 7.07 | 0.19 | 0.08 |
Spleen | 55.71 | 4.37 | 46.00 | 15.00 | 41.84 | 23.44 | 0.20 | 0.15 |
Pancreas | 1.36 | 0.43 | 1.31 | 0.83 | 0.85 | 0.36 | 0.14 | 0.09 |
Kidney | 1.45 | 0.38 | 1.14 | 0.22 | 3.74 | 3.36 | 0.44 | 0.27 |
Upper stomach | 0.94 | 0.57 | 0.44 | 0.17 | 0.53 | 0.12 | 0.24 | 0.13 |
Lower stomach | 1.35 | 0.65 | 0.77 | 0.34 | 1.01 | 0.56 | 0.31 | 0.16 |
Upper intestine | 1.45 | 0.56 | 1.26 | 0.55 | 1.23 | 0.59 | 0.12 | 0.08 |
Middle intestine | 0.69 | 0.17 | 0.46 | 0.20 | 0.58 | 0.11 | 0.12 | 0.07 |
Lower intestine | 0.59 | 0.20 | 0.39 | 0.17 | 0.70 | 0.31 | 0.11 | 0.06 |
Heart | 0.74 | 0.07 | 0.72 | 0.31 | 0.77 | 0.51 | 0.25 | 0.20 |
Lung | 1.22 | 0.52 | 1.39 | 0.75 | 1.27 | 0.65 | 0.40 | 0.29 |
Tongue | 0.85 | 0.31 | 0.87 | 0.28 | 0.64 | 0.45 | 0.18 | 0.09 |
Brain | 0.07 | 0.02 | 0.06 | 0.02 | 0.07 | 0.04 | 0.03 | 0.02 |
2A4 | 7D8 | 8G9 | MOPC 141 | |
---|---|---|---|---|
Muscle | 1.0 | 1.0 | 1.0 | 1.0 |
Skin | 1.2 | 1.1 | 1.0 | 1.4 |
Liver | 128.2 | 125.9 | 142.9 | 3.8 |
Spleen | 232.1 | 255.6 | 261.5 | 4.0 |
Pancreas | 5.7 | 7.3 | 5.3 | 2.8 |
Kidney | 6.0 | 6.3 | 23.4 | 8.8 |
Upper stomach | 3.9 | 2.4 | 3.3 | 4.8 |
Lower stomach | 5.6 | 4.3 | 6.3 | 6.2 |
Upper intestine | 6.0 | 7.0 | 7.7 | 2.4 |
Middle intestine | 2.9 | 2.6 | 3.6 | 2.4 |
Lower intestine | 2.5 | 2.2 | 4.4 | 2.2 |
Heart | 3.1 | 4.0 | 4.8 | 5.0 |
Lung | 5.1 | 7.7 | 7.9 | 8.0 |
Tongue | 3.5 | 4.8 | 4.0 | 3.6 |
Brain | 0.3 | 0.3 | 0.4 | 0.6 |
The amyloidogenic SAA protein precursor is a 103 amino acid, acute phase reactant lipoprotein synthesized in the liver; when isolated from amyloid deposits, it has been cleaved proteolytically by cathepsin-like enzymes into N-terminal fragments consisting predominantly of ∼75 residues (Yamada et al.,
The fibril-related specificity of the three mAbs also was evidenced by the fact that they bound only to the truncated AA peptide found in amyloid fibrils, but not the circulating native protein; furthermore, the longer peptide, GHEDTMADQE spanning the cleavage site, did not block the interaction between the mAbs and AA amyloid adsorbed onto microplate wells or that present in tissue sections. Notably, the AA epitope recognized by mAbs 2A4, 7D8 and 8G9 located between residues 71–75 is structurally accessible, and not blocked by molecules typically associated with amyloid deposits, e.g., proteoglycans, apolipoproteins, and serum amyloid P-component (Alexandrescu,
Small animal imaging, together with micro-autoradiography, provide an exquisitely sensitive method to determine the
The highly specific targeting of amyloid by our three mAbs, along with the lack of binding to circulating SAA or healthy (amyloid-free) tissues, suggest that these radiolabeled antibodies have diagnostic utility as imaging agents in patients with AA amyloidosis. Additionally, given that other fibril-specific mAbs have been shown to opsonize and effect removal of pathologic light chain or Aβ amyloid (Bard et al.,
Robin Barbour, Peter Seubert, and Dale Schenk are employes of Elan Pharmaceuticals.
This work was supported by a collaborative research grant from Elan Pharmaceuticals, South San Francisco, CA, USA. Alan Solomon is an American Cancer Society Clinical Research Professor.