Protection against tumors resistant to Her-2 antibody and receptor tyrosine kinase inhibitor by Her-2 DNA vaccination
Authors: Paula Whittington, Marie Piechocki, Henry Heng, Jennifer Jacob, Richard Jones, Wei-Zen Wei
Journal: Cancer Research (2008)

2852   Her-2/neu +  tumor cells resistant to antibody (Ab) and/or receptor tyrosine kinase inhibitor (RTKI) are emerging in treated patients. To investigate if such resistant tumor cells can be controlled by active vaccination, four BALB/c mouse mammary tumor (MMT) lines expressing rat neu were tested. TUBO and Bam1a were established from spontaneous tumors of neu transgenic mice. Bam IR-5 (IR-5) was a gefitinib resistant variant derived from Bam1a (1a) and D2F2/neu was a hormone induced mammary tumor transfected with neu. Treatment of TUBO and 1a with gefitinib or anti-neu Ab reduced cell proliferation and the phosphorylation of Akt. Gefitinib had no effect on IR-5 or D2F2/neu proliferation or signaling. Anti-neu monoclonal Ab showed modest inhibition of IR-5, but had no effect on D2F2/neu. Therefore, TUBO and 1a are sensitive to Ab and RTKI. IR-5 is resistant to RTKI, and less sensitive to Ab. D2F2/neu is resistant to both.  A comprehensive view of the accumulated genomic alterations in neu expressing cells was obtained by spectral karyotyping. Although TUBO and 1a are of similar origin significant disparities exist within their karyotypes. TUBO and 1a had an average of 57 and 52 chromosomes, respectively, compared to the normal 42 in murine cells. Translocations, t(15; 17), t(4; 10), t(4, 13), t(4; 10), were found in TUBO cells, not 1a. Non-Clonal Chromosomal Aberrations (NCCAs) were found at 22% frequency in TUBO, not 1a. IR-5 had 72 chromosomes with 15% NCCAs and many abnormal centromeres. IR-5 exhibited trisomies of chromosomes 6, 13 and 16, and monosomies of 18 and 19, which were absent in 1a. D2F2/neu and D2F2 cells share t(11;16) and chromosome fragments X, 13, 3 and 1. But the transfected D2F2/neu cells exhibited a more stable karyotype suggesting the selection of a less aberrant variant which expressed neu at a high level.  Vaccination with DNA encoding the extracellular and transmembrane domains of neu induced Ab and T cell responses that completely protected mice from these four tumors. After T cell depletion, protection against IR-5 and D2F2/neu was diminished 60% and 30% respectively, yet protection from TUBO and 1a tumors was not tempered. These results demonstrate the important role of T cells in the rejection of drug and Ab resistant tumor cells.  To assess if CTLs could control these tumors, in the absence of anti-neu antibodies, mice were immunized with pcytneu, which encodes a full length neu that is released into the cytoplasm and rapidly degraded by the proteasome. Anti-neu Abs were not detected in pcytneu vaccinated mice while an average of 154 + 60/10 6  neu reactive IFN-γ secreting splenocytes were detected by ELISPOT. All tumors were rejected in pcytneu immunized mice, further supporting the therapeutic effect of vaccine induced T cells. Vaccination of patients undergoing Ab or RTKI therapy may prevent disease relapse by eliminating drug resistant cells. CA76340, 125680, GM 58905-7