A single-chain IL-12 IgG3 antibody fusion protein retains antibody specificity and IL-12 bioactivity and demonstrates antitumor activity.
IL-12 is a heterodimeric cytokine with many actions on innate and cellular immunity that may have antitumor and antimetastatic effects. However, systemic administration of IL-12 can be toxic. Tumor-specific Abs provide a means to selectively target a metastatic/residual nodule and deliver therapeutic quantities of an immunostimulatory molecule like IL-12 with lower systemic levels and ideally, toxicity. We report the construction and characterization of an Ab fusion protein in which single-chain murine IL-12 is fused to an anti-Her2/neu Ab at the amino terminus (mscIL-12.her2.IgG3). The use of single-chain IL-12 in the fusion protein simplifies vector construction, ensures equimolar concentrations of the two IL-12 subunits, and may confer greater stability to the fusion protein. SDS-PAGE analysis shows this 320-kDa protein is secreted and correctly assembled. FACS analysis demonstrates that this fusion protein binds to cells transfected with the Her2/neu Ag, thus retaining Ab specificity; this fusion protein also binds to a cell line and to PHA-activated PBMC that express the IL-12R, thus demonstrating cytokine receptor specificity. T cell proliferation assays and NK cytotoxicity assays demonstrate that this fusion protein exhibits IL-12 bioactivity comparable to recombinant murine IL-12. In vivo studies demonstrate that this fusion protein has antitumor activity. These results are significant and suggest that this IL-12 Ab fusion protein can effectively combine the therapeutic potential of IL-12 with the tumor-targeting ability of the Ab and may provide a viable alternative to systemic administration of IL-12.
Peng
L S
LS
Department of Microbiology, Immunology and Molecular Genetics, Molecular Biology Institute, University of California, Los Angeles 90095, USA.
Penichet
M L
ML
Morrison
S L
SL
D000230:Adenocarcinoma / Q000276:immunology / Q000628:therapy; D000276:Adjuvants, Immunologic / Q000494:pharmacology; D000818:Animals; D000918:Antibody Specificity / Q000235:genetics*; D000951:Antigens, Neoplasm / Q000378:metabolism; D000970:Antineoplastic Agents / Q000008:administration & dosage / Q000737:chemistry* / Q000494:pharmacology; D001666:Binding Sites, Antibody / Q000235:genetics; D003110:Colonic Neoplasms / Q000276:immunology / Q000628:therapy; D003602:Cytotoxicity, Immunologic / Q000276:immunology; D005260:Female; D006801:Humans; D007074:Immunoglobulin G / Q000008:administration & dosage / Q000235:genetics*; D007279:Injections, Subcutaneous; D018664:Interleukin-12 / Q000008:administration & dosage / Q000235:genetics* / Q000378:metabolism; D020014:K562 Cells; D007694:Killer Cells, Natural / Q000276:immunology; D007963:Leukocytes, Mononuclear / Q000276:immunology; D008213:Lymphocyte Activation / Q000276:immunology; D051379:Mice; D008807:Mice, Inbred BALB C; D011485:Protein Binding / Q000235:genetics / Q000276:immunology; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000008:administration & dosage / Q000235:genetics; D011947:Receptors, Antigen, B-Cell / Q000235:genetics / Q000378:metabolism; D018123:Receptors, Interleukin / Q000378:metabolism; D053707:Receptors, Interleukin-12; D011993:Recombinant Fusion Proteins / Q000008:administration & dosage / Q000096:biosynthesis / Q000138:chemical synthesis* / Q000276:immunology*; D014407:Tumor Cells, Cultured
D000276:Adjuvants, Immunologic; D000951:Antigens, Neoplasm; D000970:Antineoplastic Agents; D007074:Immunoglobulin G; D018664:Interleukin-12; D018719:Erb-b2 Receptor Tyrosine Kinases; D011947:Receptors, Antigen, B-Cell; D018123:Receptors, Interleukin; D053707:Receptors, Interleukin-12; D011993:Recombinant Fusion Proteins