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 c ells to ErbB-2 have been isolated from cancer patients, indicating ErbB-2 a s 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 v accines. To eliminate tyrosine kinase activity, the ATP binding lysine resi due 753 was substituted with alanine by replacing codon AAA with GCA in mut ant ErbB-2A, To direct recombinant ErbB-2 to the cytoplasm where major hist ocompatibility complex (MHC) I peptide processing takes place, the endoplas mic reticulum (ER) signal sequence was deleted in cyt ErbB-2. The third con struct cyt ErbB-2A contained cytoplasmic ErbB-2 with the K to A mutation. E xpression of recombinant proteins was measured by flow cytomety in transfec ted murine mammary tumor cell line D2F2, Transmembrane ErbB-2 and ErbB-2A w ere readily detected. Cytoplasmic ErbB-2 and ErbB-2A were detected only aft er the transfected cells were incubated overnight with a proteasome inhibit or, indicating prompt degradation upon synthesis, ErbB-2 autophosphorylatio n was eliminated by the K to A mutation as demonstrated by Western blot ana lysis. Growth of ErbB-2-positive tumor in BALB/c mice was inhibited after v accination 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. (C) 1999 Wiley-Liss, Inc.