The purpose of this study was to engineer a bivalent single-chain anticarcinoembryonic
antigen (CEA) antibody and an
interleukin 2 (IL-2) fusion
protein derivative for selective
tumor targeting of
cytokines. The variable domains of a high affinity anti-CEA antibody, T84.66, were used to form a single-gene-encoded antibody [
single-chain variable fragment joined to the crystallizable
fragment, Fc (scFvFc)]. The fusion
protein (scFvFc.IL-2) consisted of mouse IL-2-fused to the COOH-terminal end of the scFvFc. The engineered
proteins were assembled as complete molecules and were similar to the intact anti-CEA
monoclonal antibody (Mab) in
antigen-binding properties. Based on
IL-2 content of the fusion
protein, its ability to support proliferation of CTLL-2 cells was identical with that of
IL-2. Despite a molecular size similar to that of the intact Mab, the blood clearance of the fusion
protein was markedly faster than that of the intact Mab or scFvFc. Incubation of radiolabeled scFvFc.IL-2 but not the intact or scFvFc
antibodies in mouse serum was accompanied by the appearance of complexes, suggesting that the latter may contribute to the accelerated clearance of the fusion
protein. Biodistribution and
tumor targeting studies were carried out in CEA-transgenic mice bearing CEA-positive murine
tumors as well as the
antigen-negative parental
tumor. The bivalent anti-CEA scFvFc had
tumor localization properties similar to those of the intact Mab. Although fusion of
IL-2 to the COOH-terminal end of the bivalent scFvFc altered its pharmacokinetic properties, the fusion antibody was able to target
tumors specifically. Maximum uptake of the intact Mab, scFvFc, and scFvFc.IL-2 in CEA-positive
tumors was 29.3 +/- 5.0, 19.5 +/- 2.1, and 6.6 +/- 0.9% injected dose/g, respectively. Maximum
tumor localization ratios (CEA-positive/CEA-negative
tumor) were similar for all three antibody types (4.6-6.0), demonstrating the
antigen specificity of the
tumor targeting. Significant
antigen-specific targeting to CEA-positive normal tissues of transgenic mice was not observed. Although the
tumor-targeting properties of the fusion
protein were low, the growth of CEA-expressing (P = 0.01) but not
antigen-irrelevant (P = 0.22) syngeneic
tumor cells was inhibited
after treatment of transgenic mice with the anti-CEA-IL-2 antibody.
Therapy of CEA-expressing
tumors was improved after i.v. administration of the fusion
protein (P = 0.0001). These studies indicate that anti-CEA antibody-directed
cytokine targeting may offer an effective treatment for CEA-expressing
carcinomas. The availability of an immunocompetent CEA transgenic mouse model will also help to determine the immunotherapeutic properties of these fusion
proteins.