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99mTc-Labeled anti-receptor for advanced glycation endproducts monoclonal antibody F(ab’)2 fragments

99mTc-anti-RAGE mF(ab’)2
, PhD
National Center for Biotechnology Information, NLM, NIH
Corresponding author.

Created: ; Last Update: August 16, 2010.

Chemical name:99mTc-Labeled anti-receptor for advanced glycation endproducts monoclonal antibody F(ab’)2 fragmentsImage RAGE.jpg
Abbreviated name:99mTc-anti-RAGE mF(ab’)2
Synonym:99mTc-anti-RAGE F(ab’)2
Agent Category:Antibodies
Target:Receptor for advanced glycation endproducts (RAGE)
Target Category:Receptors
Method of detection:Single-photon emission computed tomography (SPECT)
Source of signal / contrast:99mTc
Activation:No
Studies:
  • Checkbox In vitro
  • Checkbox Rodents
Structure of RAGE.

Background

[PubMed]

The 99mTc-labeled anti-receptor for advanced glycation endproducts (RAGE) monoclonal antibody F(ab’)2 fragment, abbreviated as 99mTc-anti-RAGE mF(ab’)2 or 99mTc-anti-RAGE F(ab’)2, is a radiotracer developed by Tekabe et al. for imaging atherosclerotic lesions by targeting highly expressed RAGE with single-photon emission computed tomography (SPECT) (1). RAGE is a 35-kDa transmembrane receptor of the immunoglobulin (Ig) superfamily (2, 3).

RAGE has one V domain, two C domains, one transmembrane domain, and one cytoplasmic tail. The V domain consists of two N-glycosylation sites and is responsible for extracellular ligand binding. RAGE exists in three forms: full-length, membrane-bound, and soluble (2-4). Under physiological conditions, RAGE is expressed at low levels in a variety of cells in a regulated manner, but RAGE is highly expressed in a series of age- and diabetes-related chronic inflammatory diseases and cancer (5-8). The advanced glycation endproducts (AGEs) are the major ligands for RAGE. AGEs are a heterogeneous group of peptides and proteins derived from non-enzymatic glycosylation processes (3, 5). Large amounts of AGEs is formed through metabolism and aging, and this establishes a positive feedback cycle under pathological conditions such as diabetes (2, 4, 9). The interaction between AGEs and RAGE affects almost all types of cells and molecules and results in pro-inflammatory gene activation (8, 10). The pathological effects induced by the AGEs/RAGE interaction include increasing vascular permeability, inhibiting vascular dilation, inducing cytokine secretion, enhancing oxidative stress, and modulating cell response to exogenous growth factors. Understanding of the AGEs/RAGE interaction is crucial to develop new treatment regimens for age- and diabetes-related conditions and cancer (4, 10).

In the case of atherosclerosis, AGEs are formed in both diabetic and nondiabetic conditions, but to a greater extent in diabetes. RAGE itself is expressed in nearly all cell types pertinent for the development and progression of atherosclerotic plaque. The AGEs/RAGE interaction leads to diabetic vascular complications and augments atherosclerotic plaque development and progression (8, 9). In a proof-of-concept study of whether the expression level of RAGE could be detected and thus used as a marker of atherosclerosis, Tekabe et al. developed a 99mTc-labeled polyclonal antibody F(ab’)2 fragment against RAGE and further demonstrated that uptake of the fragment in the atherosclerotic plaques could be visualized with planar γ imaging in a nondiabetic apolipoprotein E–null (ApoE–/–) mouse model (2). To reduce the nonspecific binding associated with the polyclonal antibody, Tekabe et al. developed a monoclonal antibody against the extracellular domain of RAGE (1). The intact monoclonal antibody was then digested with pepsin, and the generated F(ab’)2 fragment was radiolabeled to produce the radiotracer 99mTc-anti-RAGE mF(ab’)2. SPECT imaging showed that the radiotracer identified early accelerated lesions in diabetic ApoE–/– mice to a better degree than in nondiabetic mice. The results indicated that SPECT with 99mTc-anti-RAGE mF(ab’)2 may be used to assess novel therapies in experimental animals and possibly in humans (1).

Synthesis

[PubMed]

Tekabe et al. described in detail the production and radiolabeling of the monoclonal F(ab’)2 fragment (1). Briefly, the peptide Ac-NRRGKEVKSNYRVRVYQIC-amide on the basis of the V domain of RAGE was synthesized and conjugated to the carrier molecule keyhole limpet hemocyanin (1, 2). After immunization of the BALB/c mice (n = 15) with the conjugated peptide, hybridomas were selected, and monoclonal antibodies were then produced in vitro (<3 endotoxin U/mg purified antibody). The F(ab')2 fragments were generated with pepsin digestion of the intact antibody. 99mTc-Labeling of the fragments was performed with diethylenetriamine pentaacetic acid (DTPA) modification, followed by reaction with 99mTcO4- in SnCl2 in 0.1 N HCl. The specific activity of the 99mTc-anti-RAGE mF(ab’)2 was 6.58 ± 0.68 MBq/µg (0.178 ± 0.018 mCi/µg) protein, and the radiochemical purity was 98 ± 0.54%. The molecular weight of the fragments and the radiolabeling yields were not reported (1).

In Vitro Studies: Testing in Cells and Tissues

[PubMed]

Immunoreactivities of the intact antibody and antibody fragment were tested against soluble RAGE (1). The antibody concentration for the DTPA-modified F(ab')2 fragment and the unmodified intact antibody, given 50% of maximum binding, was 0.2 µg/ml and 0.4 µg/ml, respectively, which was equivalent to 2 × 10-9 mol/L or apparent affinity of 0.5 × 109 L/mol for both intact antibody and antibody fragment. The results suggested no reduction of the binding capability for the DTPA-modified F(ab')2 fragment compared with the unmodified intact antibody.

Animal Studies

Rodents

[PubMed]

Tekabe et al. measured the blood-pool clearance of 99mTc-anti-RAGE mF(ab’)2 in C57BL/6 mice (n = 3) to determine the optimal time for imaging (1). The clearance was biexponential, with the half-life equal to 20 min for the first (fast) component and 7 h for the second (slower) component. The optimal time for imaging was 4 h after tracer injection.

For biodistribution and imaging studies, Tekabe et al. established animal models of atherosclerosis with male diabetic and nondiabetic ApoE–/– mice (1). Diabetes was induced in the mice via five daily intraperitoneal injections of streptozotocin (resulting in insulin deficiency). Male ApoE–/–/RAGE–/– mice were used as controls. Biodistribution studies were performed 5–6 h after injection of the 99mTc-anti-RAGE mF(ab’)2, and the highest uptake was observed in the liver and spleen in both diabetic and nondiabetic ApoE–/– mice (n = 6–8/group). SPECT imaging at 4 h after injection showed uptake in the atherosclerotic lesions of the proximal aorta from both diabetic and nondiabetic ApoE–/– mice (n = 8/group). However, the uptake was significantly greater in the diabetic group (1.39 × 10–2 ± 0.16% injected dose (ID)) than in the nondiabetic group (0.48 × 10–2 ± 0.27% ID; P < 0.0001). The control ApoE–/–/RAGE–/– mice (n = 6) showed no uptake of the radiotracer. The nondiabetic ApoE–/– mice showed minimal uptake of the radiotracer; the significance was borderline (P = 0.05) in comparison to the ApoE–/–/RAGE–/– mice. Atherosclerotic ApoE–/– mice injected with an irrelevant 99mTc-labeled antibody fragment as a control showed no tracer uptake in the atherosclerotic plaque. No blocking studies were presented. The concentration of the potential competitor, AGE, for the binding site was not determined and this topic was not considered further.

Ex vivo radioactivity counting confirmed the uptake of 99mTc-anti-RAGE mF(ab’)2 in the proximal aortic atherosclerotic lesion, showing a close correlation for the uptakes from in vivo SPECT scans and from ex vivo counts (r = 0.82; P = 0.0001). A significant difference was also obtained for the radioactivity count between the diabetic and nondiabetic ApoE–/– mice (0.36 ± 0.19% ID versus 0.052 ± 0.028% ID per gram tissue; P = 0.002). Histology of the aortic sections confirmed the formation and severity of the lesions as American Heart Association class III lesions in diabetic ApoE–/– mice, class II lesions in nondiabetic ApoE–/– mice, and minimal lesions in control ApoE–/–/RAGE–/– mice.

Tekabe et al. further performed a correlative analysis between radiotracer uptake and RAGE expression (1). With immunohistochemical staining, the percentage of RAGE-positive cells in the proximal aortic lesions was more than two-fold higher in diabetic mice (37.7 ± 6.9%) than in nondiabetic mice (15.9 ± 4.7%; P = 0.0008). Total macrophage burden in the lesions was also significantly higher in the diabetic mice (28.2 ± 7.6%) than in the nondiabetic mice (13.4 ± 4.8%; P = 0.0004). RAGE was colocalized predominantly with macrophages. The radiotracer uptake correlated strongly with the RAGE expression (r = 0.82; P = 0.002) and with the macrophage burden (r = 0.86; P < 0.0001). The specificity of the anti-RAGE antibody was further confirmed by the lack of staining in the aortic lesions in the control ApoE–/–/RAGE–/– mice.

Other Non-Primate Mammals

[PubMed]

No references are currently available.

Non-Human Primates

[PubMed]

No references are currently available.

Human Studies

[PubMed]

No references are currently available.

References

1.
Tekabe Y., Luma J., Einstein A.J., Sedlar M., Li Q., Schmidt A.M., Johnson L.L. A novel monoclonal antibody for RAGE-directed imaging identifies accelerated atherosclerosis in diabetes. J Nucl Med. 2010;51(1):92–7. [PubMed: 20008983]
2.
Tekabe Y., Li Q., Rosario R., Sedlar M., Majewski S., Hudson B.I., Einstein A.J., Schmidt A.M., Johnson L.L. Development of receptor for advanced glycation end products-directed imaging of atherosclerotic plaque in a murine model of spontaneous atherosclerosis. Circ Cardiovasc Imaging. 2008;1(3):212–9. [PubMed: 19808545]
3.
Yan S.F., Ramasamy R., Schmidt A.M. The RAGE axis: a fundamental mechanism signaling danger to the vulnerable vasculature. Circ Res. 2010;106(5):842–53. [PMC free article: PMC2862596] [PubMed: 20299674]
4.
Sourris K.C., Forbes J.M. Interactions between advanced glycation end-products (AGE) and their receptors in the development and progression of diabetic nephropathy - are these receptors valid therapeutic targets. Curr Drug Targets. 2009;10(1):42–50. [PubMed: 19149535]
5.
Daroux M., Prevost G., Maillard-Lefebvre H., Gaxatte C., D'Agati V.D., Schmidt A.M., Boulanger E. Advanced glycation end-products: implications for diabetic and non-diabetic nephropathies. Diabetes Metab. 2010;36(1):1–10. [PubMed: 19932633]
6.
Abe R., Yamagishi S. AGE-RAGE system and carcinogenesis. Curr Pharm Des. 2008;14(10):940–5. [PubMed: 18473843]
7.
Fang F., Lue L.F., Yan S., Xu H., Luddy J.S., Chen D., Walker D.G., Stern D.M., Schmidt A.M., Chen J.X., Yan S.S. RAGE-dependent signaling in microglia contributes to neuroinflammation, Abeta accumulation, and impaired learning/memory in a mouse model of Alzheimer's disease. FASEB J. 2010;24(4):1043–55. [PMC free article: PMC3231946] [PubMed: 19906677]
8.
Nienhuis H.L., Westra J., Smit A.J., Limburg P.C., Kallenberg C.G., Bijl M. AGE and their receptor RAGE in systemic autoimmune diseases: an inflammation propagating factor contributing to accelerated atherosclerosis. Autoimmunity. 2009;42(4):302–4. [PubMed: 19811283]
9.
Walcher D., Marx N. Advanced glycation end products and C-peptide-modulators in diabetic vasculopathy and atherogenesis. Semin Immunopathol. 2009;31(1):103–11. [PubMed: 19347338]
10.
Ramasamy R., Yan S.F., Schmidt A.M. RAGE: therapeutic target and biomarker of the inflammatory response--the evidence mounts. J Leukoc Biol. 2009;86(3):505–12. [PubMed: 19477910]

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