Protection against mammary tumor growth by vaccination with full-length, modified human ErbB-2 DNA.
ErbB-2 is overexpressed in several human cancers and conveys a transforming activity that is dependent on tyrosine kinase activity. Antibodies and T cells to ErbB-2 have been isolated from cancer patients, indicating ErbB-2 as a potential target of active vaccination. In this study, 3 mutant ErbB-2 DNA constructs encoding full-length, ErbB-2 proteins were tested as tumor vaccines. To eliminate tyrosine kinase activity, the ATP binding lysine residue 753 was substituted with alanine by replacing codon AAA with GCA in mutant ErbB-2A. To direct recombinant ErbB-2 to the cytoplasm where major histocompatibility complex (MHC) I peptide processing takes place, the endoplasmic reticulum (ER) signal sequence was deleted in cyt ErbB-2. The third construct cyt ErbB-2A contained cytoplasmic ErbB-2 with the K to A mutation. Expression of recombinant proteins was measured by flow cytometry in transfected murine mammary tumor cell line D2F2. Transmembrane ErbB-2 and ErbB-2A were readily detected. Cytoplasmic ErbB-2 and ErbB-2A were detected only after the transfected cells were incubated overnight with a proteasome inhibitor, indicating prompt degradation upon synthesis. ErbB-2 autophosphorylation was eliminated by the K to A mutation as demonstrated by Western blot analysis. Growth of ErbB-2-positive tumor in BALB/c mice was inhibited after vaccination with ErbB-2 or ErbB-2A, but not with cyt ErbB-2 or cyt ErbB-2A. ErbB-2A that is free of tyrosine kinase activity is a potential candidate for anticancer vaccination. The 3 mutant constructs should be useful tools to delineate the role of individual immune effector cell in ErbB-2-specific antitumor immunity and to develop strategies for enhancing such immunity.
D019943:Amino Acid Substitution; D000818:Animals; D019496:Cancer Vaccines / Q000235:genetics / Q000627:therapeutic use*; D002455:Cell Division / Q000187:drug effects / Q000276:immunology; D005260:Female; D005434:Flow Cytometry; D015870:Gene Expression; D018734:Genes, erbB-2 / Q000235:genetics / Q000276:immunology*; D006801:Humans; D008325:Mammary Neoplasms, Experimental / Q000235:genetics / Q000628:therapy*; D051379:Mice; D008807:Mice, Inbred BALB C; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D010957:Plasmids / Q000235:genetics / Q000276:immunology; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D017384:Sequence Deletion; D014162:Transfection; D014407:Tumor Cells, Cultured; D014443:Tyrosine / Q000378:metabolism; D019444:Vaccines, DNA / Q000235:genetics / Q000627:therapeutic use*