Generation and characterization of a single-gene encoded single-chain immunoglobulin-interleukin-2 fusion protein.
BACKGROUND
Interleukin-2 (IL-2), a potent inducer of cellular immune responses, has been used for biological therapy of human cancer; however, the high doses of IL-2 required to mediate patients' immune responses can cause considerable systemic toxicity. The murine monoclonal antibody (MAb) CC49, which reacts with tumor-associated glycoprotein (TAG)-72, expressed on a variety of human carcinomas, has shown excellent tumor localization in recent clinical trials.


OBJECTIVE
Development and characterization of a single-chain immunoglobulin-IL-2 (SCIg-IL-2) fusion protein which, by delivering IL-2 selectively to the tumor site, can serve as an effective reagent for CC49/IL-2 combination therapy.


METHODS
A single-gene encoding the SCIg-IL-2 fusion protein derived from the chimeric (c) CC49 was designed, generated and inserted in an expression vector. The monomeric single-chain protein consisted of the CC49 heavy and light chain variable domains covalently jointed through a (GGGGS)3 linker peptide. The carboxyl end of the variable domain of the light chain was linked to the amino terminus of the human gamma 1 Fc through the hinge region, and the carboxyl end of the CH3 domain was linked to the amino terminus of the human IL-2 through a GGGSGGG linker peptide. The SCIg-IL-2, expressed from the murine myeloma cells transfected with the expression construct, was characterized for its antigen-binding specificity, antibody effector functions and IL-2 biological activity.


CONCLUSIONS
Transfection of murine myeloma cells with the single-gene expression construct SCIg-IL-2 expressed a single-chain protein of approximately 70 kD, which was secreted into tissue culture fluid as a homodimer of approximately 140 kD. SCIg-IL-2 competed completely with cCC49 for binding to the TAG-72 antigen, but approximately three- to four-fold more of the SCIg-IL-2 was required to achieve levels of competition similar to those observed with the murine or chimeric CC49. With human effector cells, the fusion protein mediated lysis of TAG-72-positive human carcinoma cells. Prior treatment of human effector cells with 100 U/ml of human IL-2 enhanced the fusion protein-mediated cytolysis from 32 to 65%. At doses of > or = 1 ng/ml, the stimulatory effect of SCIg-IL-2 on IL-2 dependent murine HT-2 cell proliferation was comparable to that of the recombinant human IL-2. The single-gene construct may also facilitate inoculation of the gene in animal tissue for in vivo expression of the fusion protein.
Shu
L
L
Laboratory of Tumor Immunology and Biology, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892-1750, USA.
Qi
C F
CF
Hand
P H
PH
Schlom
J
J
Kashmiri
S V
SV
D000911:Antibodies, Monoclonal; D019281:Dimerization; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007111:Immunity, Cellular; D007118:Immunoassay; D007141:Immunoglobulin Fc Fragments / Q000008:administration & dosage / Q000235:genetics / Q000276:immunology; D007376:Interleukin-2 / Q000008:administration & dosage / Q000235:genetics* / Q000276:immunology; D009101:Multiple Myeloma / Q000378:metabolism; D011485:Protein Binding; D015202:Protein Engineering; D011993:Recombinant Fusion Proteins / Q000008:administration & dosage / Q000235:genetics* / Q000276:immunology; D014407:Tumor Cells, Cultured
D000911:Antibodies, Monoclonal; D007141:Immunoglobulin Fc Fragments; D007376:Interleukin-2; D011993:Recombinant Fusion Proteins