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The Humoral Immune Response to Macrocyclic Chelating Agent DOTA Depends on the Carrier Molecule

Unit of Molecular Therapies, Department of Experimental Oncology, Istituto Nazionale Tumori, Milan; and Division of Nuclear Medicine, European Institute of Oncology, Milan, Italy

View larger version (34K): [in a new window]   FIGURE 1. Number of injections needed to evoke humoral anti-DOTA response in mice receiving low-dose cM19-DOTA-biotin. Antibody binding was detected by quantitative ELISA on HSA-DOTA-biotin. Sera from 4 mice, obtained 1 wk after each injection, were pooled, diluted 1:200, and tested in duplicate. Data are mean ± SD from 2 independent experiments. A = absorbance; i.p. = intraperitoneal injection; pre = before injection; s.c. = subcutaneous injection.

View larger version (28K): [in a new window]   FIGURE 2. Specificity of binding reactivity of sera from mice immunized with different DOTA carriers. Binding activity was assessed on biotinylated HSA (hatched bars) or cM19 (solid bars), conjugated (A) or not conjugated (B) to DOTA, by quantitative ELISA. Sera (diluted 1:200 and tested in duplicate) were obtained after third injection (day 35) of immunogens, as described in Table 2. Data are mean ± SD of 2 replicates. A = absorbance.

View larger version (18K): [in a new window]   FIGURE 3. Inhibition of mAb MDOTA-1 binding by sera of DOTA carrier-immunized mice. 125I-labeled MDOTA-1 and serially diluted sera (starting from 1:25) from mice immunized with low-dose TOC (), high-dose TOC (), low-dose cM19 (), high-dose cM19 (), low-dose mM19 (), and high-dose mM19 (•) were incubated on HSA-DOTA-biotin. Samples were tested in duplicate; data are mean of 2 replicates (SD is not indicated because it was smaller than symbol).

View larger version (16K): [in a new window]   FIGURE 4. Efficiency of DOTA presentation by different carriers as tested by binding inhibition of anti-DOTA mAb MDOTA-1. Mixture of 125I-labeled MDOTA-1 (2.5 x 105 cpm; 50 µL per well) and serial dilutions of DOTATOC (), cM19-DOTA-biotin (), mM19-DOTA-biotin (•), or HSA-DOTA-biotin () were incubated on HSA-DOTA-biotin. Each sample was tested in duplicate; data are mean of 2 replicates (SD is not indicated because it was smaller than symbol). Inhibitory concentrations of 50% (related to DOTA molarity) were 2.8 x 10-7 mol/L (cM19-DOTA-biotin), 4.4 x 10-7 mol/L (mM19-DOTA-biotin), 6 x 10-7 mol/L (HSA-DOTA-biotin), and 8 x 10-6 mol/L (DOTATOC). Molar excess, related to target (HSA-DOTA-biotin) molarity (2 x 10-9 mol/L), was x138 (cM19-DOTA-biotin), x220 (mM19-DOTA-biotin), x300 (HSA-DOTA-biotin), and x3,000 (DOTATOC). M = mol/L.