title: Targeting and therapy of carcinoembryonic antigen-expressing tumors in transgenic mice with an antibody-interleukin 2 fusion protein.
abstract: 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.
authors.affiliation: Department of Pathology, Vanderbilt University Medical Center, Nashville, Tennessee 37232, USA.
authors.forename: X
authors.initials: X
authors.lastname: Xu
authors.forename: P
authors.initials: P
authors.lastname: Clarke
authors.forename: G
authors.initials: G
authors.lastname: Szalai
authors.forename: J E
authors.initials: JE
authors.lastname: Shively
authors.forename: L E
authors.initials: LE
authors.lastname: Williams
authors.forename: Y
authors.initials: Y
authors.lastname: Shyr
authors.forename: E
authors.initials: E
authors.lastname: Shi
authors.forename: F J
authors.initials: FJ
authors.lastname: Primus
mesh_terms: D000818:Animals; D002272:Carcinoembryonic Antigen / Q000276:immunology* / Q000378:metabolism; D003110:Colonic Neoplasms / Q000188:drug therapy / Q000276:immunology / Q000378:metabolism; D004355:Drug Stability; D005260:Female; D005818:Genetic Engineering; D007128:Immunoglobulin Fragments / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D000922:Immunotoxins / Q000235:genetics / Q000493:pharmacokinetics / Q000494:pharmacology*; D007376:Interleukin-2 / Q000493:pharmacokinetics / Q000494:pharmacology*; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D009368:Neoplasm Transplantation; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000493:pharmacokinetics / Q000494:pharmacology*; D014018:Tissue Distribution
chemical_list: D002272:Carcinoembryonic Antigen; D007128:Immunoglobulin Fragments; D000922:Immunotoxins; D007376:Interleukin-2; D011993:Recombinant Fusion Proteins