Differences in binding of (99m)Tc-disintegrins to integrin alphavbeta3 on tumor and vascular cells

Abstract

Disintegrins, which contain an Arg-Gly-Asp sequence in their binding domains are antagonists of integrins such as alphavbeta3. The purpose of this study was to compare a range of disintegrins with different integrin selectivities for their binding behavior in vitro to vascular endothelial cells bearing alphavbeta3 and to cultured tumor cells which express alphavbeta3.

Methods: Five disintegrins (bitistatin, kistrin, flavoridin, VLO4 and echistatin) and a cyclic pentapeptide, c[RGDyK], were radiolabeled with (99m)Tc and tested for binding to cells in vitro.

Results: (99m)Tc-Kistrin, flavoridin and VLO4 had the highest binding, (99m)Tc-echistatin had moderate binding, and (99m)Tc-bitistatin and (99m)Tc-c[RGDyK] had low binding to cells. The observed binding was attributed to alphavbeta3 to various extents: echistatin, bitistatin>kistrin>flavoridin>VLO4. Cancer cells internalized bound disintegrins after binding, but endothelial cells did not. After binding to endothelial cells, (99m)Tc-kistrin was not displaced by competing peptide or plasma proteins.

Conclusions: These data suggest that radiolabeled kistrin, flavoridin and VLO4 may have advantages over labeled bitistatin and small cyclic peptides for targeting alphavbeta3 in vivo. Since receptor-bound radioligand is not internalized by endothelial cells, disintegrins may provide an advantage for targeting alphavbeta3 on vasculature because they bind strongly to surface receptors and are not readily displaced.

Figure 1. Specificity of Binding of ^99mTc-ligands to HUVEC

Radioligands (2 nM) were allowed to bind to cells for 1 hour before separating cells from supernatant. Black bars: Total binding. White bars: Nonspecific Binding (in the absence of divalent cations). Grey bars: Binding in the presence of excess c[RGDfV] peptide. Data represent the mean of independent experiments. Error bars = 1 sd. * Total binding > echistatin, bitistatin, c[RGDyK] (P<0.05) ** Total binding > flavoridin, bitistatin, c[RGDyK] (P<0.05) *** Total binding > bitistatin, c[RGDyK] (P<0.05)

Figure 2. Specificity of Binding of ^99mTc-ligands to SK-MEL-28 Melanoma cells

Radioligands (2 nM) were allowed to bind to cells for 1 hour before separating cells from supernatant. Black bars: Total binding. White bars: Nonspecific Binding (in the absence of divalent cations). Grey bars: Binding in the presence of excess c[RGDfV] peptide. Data represent the mean of independent experiments. Error bars = 1 sd. * Total binding > all others (P<0.05) ** Total binding > VLO4, echistatin, bitistatin, c[RGDyK] (P<0.05) *** Total binding > echistatin, bitistatin, c[RGDyK] (P<0.05) **** Total binding > bitistatin (P<0.05)

Figure 3. Specificity of Binding of ^99mTc-ligands to MDA-MB-435S breast cancer cells

Radioligands (2 nM) were allowed to bind to cells for 1 hour before separating cells from supernatant. Black bars: Total binding. White bars: Nonspecific Binding (in the absence of divalent cations). Grey bars: Binding in the presence of excess c[RGDfV] peptide. Data represent the mean of independent experiments. Error bars = 1 sd. * Total binding > VLO4, kistrin, echistatin, bitistatin, c[RGDyK] (P<0.05) ** Total binding > echistatin, bitistatin, c[RGDyK] (P<0.05) *** Total binding > bitistatin (P<0.05) **** Total binding > c[RGDyK] (P<0.05)

Figure 4

Test of internalization of ^99mTc-kistrin by MDA-MB-435S cells and HUVEC. Black bars = total radioligand binding. Gray bars = internalized radioligand. Open bars = nonspecific binding. Data represent the mean of 3 replicates. Error bars = 1 sd.

Figure 5

Internalization of bound ^99mTc-ligands to cells at 37°C after 1 hour of incubation. Open bars = % of added ligand specifically bound to cells (Total binding – nonspecific binding). Shaded bars = % of added ligand that was internalized. A. ^99mTc-bitistatin. B. ^99mTc-kistrin. C. ^99mTc-VLO4. D. ^99mTc-echistatin. E. ^99mTc-flavoridin. F. ^99mTc-c[RGDyK]. Data represent the mean of independent experiments. Error bars = 1 s.d.

Figure 6

Addition of competing ligand causes increased binding, not displacement, of radiolabeled kistrin. After radiolabeled kistrin was allowed to bind to cells for 1 hour at 37°C, excess competing ligand was added and the binding was assessed over the next hour. Binding before addition of competing ligand is represented by dashed line. A. ^99mTc-kistrin binding to MVEC, challenged with c[RGDfV] (final concentration 8 μM). B. ¹²⁵I-kistrin binding to HUVEC, challenged with heparinized plasma (filled diamonds) or binding buffer (open diamonds).