99mTc-Type II tumor necrosis receptor-Fc-interlukin-1 receptor antagonist fusion protein


Leung K.

Publication Details



In vitro Rodents



Tumor necrosis factor (TNF) is a proinflammatory cytokine produced by macrophages, and it plays an important role in inflammation and immune response (1). There are two types of TNF (TNF-α, TNF-β). TNF-α is mainly responsible for initiating inflammatory responses by induction of several proinflammatory cytokines, chemokines, matrix metalloproteinases, and vascular endothelial adhesion molecules that attract leukocytes known to promote inflammation. There are two types of TNF receptors: TNFR1 and TNFR2. Soluble forms of TNF receptors are released upon proteolytic cleavage of the membrane-bound TNF receptors. Soluble TNF receptors participate in limiting the availability of TNF to bind to its receptors (2, 3). Several anti-TNF monoclonal antibodies (TNF blockers) to further reduce circulating TNF have been approved by the United States Food and Drug Administration (FDA) for the treatment of a variety of inflammatory diseases (4, 5).

The interleukin-1 family consists of two proinflammatory cytokines, IL-1α and IL-1β, which bind to two IL-1 receptors (IL-1R1 and IL-1R2), and an IL-1R antagonist (IL-1ra), which is mainly produced by activated macrophages and tissue macrophages (6). IL-1α and IL-1β are important mediators of the inflammatory response and hematopoiesis, and they are involved in a variety of cellular activities, including cell proliferation, differentiation, and apoptosis. IL-1 is involved in chronic inflammatory diseases and in neuropathological conditions (7, 8). The balancing act of IL-1 and IL-1ra plays an important role in the regulation of inflammation and immune responses (9). IL-1ra has been shown to be effective as an anti-inflammatory treatment in several chronic inflammatory diseases and stroke (10, 11). A human recombinant, non-glycosylated form of the human IL-1ra (rhIL-1ra, Anakinra) has been approved by the FDA for the treatment of rheumatoid arthritis (12).

IL-1β and TNF-α exhibit additive or synergistic effects in promoting pathophysiological processes observed in many inflammatory diseases (13). A dual domain TNFR2-Fc-IL-1ra fusion protein was constructed by joining TNFR2 and IL-1ra cDNA to the Fc fragment of human IgG1 cDNA in an expression plasmid (14). The amino-terminal segment binds to TNF, and the carboxyl-terminal sequence binds to the IL-1R. Liu et al. (14) radiolabeled TNFR2-Fc-IL-1ra with 99mTc via 2-iminothiolane reduction to produce 99mTc-TNFR2-Fc-IL-1ra for use with single-photon emission computed tomography (SPECT) imaging of inflammation in mice.



Liu et al. (14) reported the synthesis of 99mTc-TNFR2-Fc-IL-1ra. TNFR2-Fc-IL-1ra was incubated with 2-iminothiolane for 30 min at 37°C in phosphate-buffered saline (PBS, pH 7.4). A solution of 925–1,110 MBq (25–30 mCi) 99mTcO4− was added to a mixture of SnCl2 and glucoheptonic acid and incubated at room temperature for 5 min to produce 99mTc-glucoheptonate, which was then incubated with the thiolated TNFR2-Fc-IL-1ra for 30 min at room temperature, with a radiolabel yield of >97%. 99mTc-TNFR2-Fc-IL-1ra was purified on a PD-10 column, with >95% radiochemical purity. The specific activity was 979–1,161 MBq/nmol (26.5–31.4 mCi/nmol). 99mTc-TNFR2-Fc-IL-1ra remained >95% intact for up to 5 h in both saline at room temperature and serum at 37°C. For comparison studies, 99mTc-IL-1ra-Fc and 99mTc-TNFR2-Fc were prepared similarly, with specific activities of 297–409 MBq/nmol (8.0–11.1 mCi/nmol) and 766–1,034 MBq/nmol (20.7–27.9 mCi/nmol), respectively.

In Vitro Studies: Testing in Cells and Tissues


Liu et al. (14) showed that 99mTc-TNFR2-Fc-IL-1ra remained >95% and ~90% at 6 h and 21 h in either saline at room temperature or rat serum at 37°C, respectively.

Animal Studies



Liu et al. (14) performed ex vivo biodistribution studies in normal rats (n = 5/group) at 3 h after intravenous injection of 130–204 MBq (3.5–5.5 mCi) 99mTc-TNFR2-Fc-IL-1ra, 99mTc-IL-1ra-Fc, or 99mTc-TNFR2-Fc. The biodistribution patterns were similar for all three radioligands, with each agent showing the highest accumulation in the kidneys (3.79%–5.18% injected dose/gram (ID/g), followed by the liver (1.29%–1.43% ID/g), small intestine (1.24%–2.39% ID/g), spleen (0.46%–0.81% ID/g), lung (0.45%–0.65% ID/g), heart (0.21%–0.42% ID/g), and stomach (0.13%–0.67% ID/g). Low radioactivity levels (<0.2% ID/g) were found in the large intestine, skin, and muscle. Blood radioactivity of 99mTc-IL-1ra-Fc (1.27 ± 0.15% ID/g) was significantly lower than that of 99mTc-TNFR2-Fc-IL-1ra (2.07 ± 0.07) and 99mTc-TNFR2-Fc (2.10 ± 0.03) (P < 0.05). 99mTc-TNFR2-Fc-IL-1ra remained >96% intact in the plasma at 3 h after injection.

Liu et al. (14) performed SPECT imaging studies in mice with TPA-induced edema in the right ears (n = 3–5/group) at 3 h after intravenous injection of 130–167 MBq (3.5–4.5 mCi) 99mTc-TNFR2-Fc-IL-1ra, 99mTc-IL-1ra-Fc, or 99mTc-TNFR2-Fc. The inflamed ears were clearly visualized, whereas the contralateral ears were almost invisible. There was less radioactive accumulation of 99mTc-IL-1ra-Fc in the inflamed ears compared to the accumulation of 99mTc-TNFR2-Fc-IL-1ra and 99mTc-TNFR2-Fc. Pretreatment with excess corresponding unlabeled ligand 30 min before the tracer reduced the radioactivity level to that of the contralateral left ears. Ex vivo studies showed that the radioactivity level of 99mTc-TNFR2-Fc-IL-1ra (7.81 ± 0.86% ID/g) in the inflamed ears was significantly higher than that of 99mTc-IL-1ra-Fc (2.76 ± 0.20% ID/g) and 99mTc-TNFR2-Fc (5.86 ± 0.40% ID/g) (P < 0.05), with inflamed ear/control ear ratios of 4.13, 4.78, and 2.31, respectively. Pretreatment with excess corresponding unlabeled ligand 30 min before the tracer reduced the ratios to 2.09, 2.32, and 1.69, respectively. The levels of IL-1β and TNF-α were significantly increased in the TPA-treated ears compared with contralateral ears (IL-1β: 424 ± 15 pg/mg protein versus 65 ± 21 pg/mg protein, P < 0.05; TNF-α: 33.7 ± 4.2 pg/mg protein versus 16.8 ± 1.3 pg/mg protein, P < 0.01).

Other Non-Primate Mammals


No publication is currently available.

Non-Human Primates


No publication is currently available.

Human Studies


No publication is currently available.

NIH Support

R01 HL090716, P41 EB002035


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