A novel heterogeneous bifunctional reagent containing an ester bond, N-[[4-(2-maleimidoethoxy)-succinyl]oxy]succinimide (MESS), was designed and synthesized for the conjugation of antibodies with the gallium-67 (67Ga) chelate of succinyldeferoxamine (SDF) via the ester bond. MESS was synthesized by the acylation of N-(2-hydroxyethyl)maleimide with succinic anhydride, followed by the activation of the resulting carboxylic acid to a succinimido ester. MESS possesses a maleimide group for protein conjugation and an active ester group for deferoxamine (DFO) coupling, and the two functional groups are linked via ester bonding. Conjugation of 67Ga-SDF with nonspecific human IgG was performed by reacting freshly thiolated IgG with the reaction product of MESS and DFO, followed by 67Ga labeling of the resulting conjugate using GaCl3 (67Ga-DFO-MESS-IgG). For comparison, 67Ga-DFO conjugated nonspecific human IgG with a nonmetabolizable linkage was synthesized under the same conjugation conditions as those for 67Ga-DFO-MESS-IgG, using a nonmetabolizable heterogenous bifunctional reagent [N-[(6-maleimidocaproyl)oxy]succinimide, EMCS] instead of MESS (67Ga-DFO-EMCS-IgG). HPLC size-exclusion chromatography of both preparations showed a single radioactivity and UV peak corresponding to the intact IgG. Generation of 67Ga-SDF from the 67Ga-DFO-MESS-IgG was demonstrated by reverse-phase HPLC analysis and cellulose acetate electrophoresis after the incubation of 67Ga-DFO-MESS-IgG in a buffered solution containing carboxyesterase. After injection of 67Ga-DFO-MESS-IgG into mice, faster radioactivity clearance from the blood and less radioactivity accumulation in the liver, kidney, and spleen was noted than when 67Ga-DFO-EMCS-IgG was injected.(ABSTRACT TRUNCATED AT 250 WORDS)

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PubMedDate not supplied

Induced immunity by expression of interleukin-2 or GM-CSF gene in murine neuroblastoma cells can generate antitumor response to established tumors.

We examined whether antitumor immunity could be generated by the inoculation of cytokine-producing murine neuroblastoma cells (C1300), and whether the immunity might be effective for the established tumors of wild-type (wt) cells. For that purpose, we transduced low immunogenic C1300 cells with interleukin-2 (IL-2), GM-CSF, or IL-4 genes. A loss of tumorigenicity in syngeneic mice was observed using IL-2- and GM-CSF- but not IL-4-producing C1300 cells, although their in vitro growth rates were not affected by the transduction. The syngeneic mice that had rejected IL-2 or GM-CSF producers did not develop tumors of wt cells inoculated subsequently, but formed tumors of irrelevant syngeneic mammary tumor cells. Accordingly, the inoculation of IL-2 or GM-CSF producers into immunocompetent mice generated tumor-specific acquired immunity. The induced immunity using IL-2 or GM-CSF producers was also effective in eradicating established subcutaneous tumors of wt cells and in reducing the number of preexisting metastatic foci in the liver. These data suggest a potential application of IL-2- or GM-CSF-producing syngeneic tumor cells for the treatment of low immunogenic neuroblastomas.

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