Figures & data
Figure 1 Tumor delay after treatment with anti-VEGFR2/Flk-1 vaccines in an autochthonous model for Her-2/neu breast cancer. Six week old mice expressing a transgene for rat Her-2/neu were vaccinated with a 1 × 108 CFU of Lm-LLO-Flk-E1, Lm-LLO-Flk-11, or a control Lm i.p. for a total of six sequential treatments. Each vaccine was given every three weeks, from six weeks and continuing to 21 weeks of age at which time mice were observed on a weekly basis for the presence of mammary tumors. Mice were followed out beyond 55 weeks. N = 15 mice per group. At 55 weeks, 0/15 mice were tumor negative for the control Lm group, 3/15 were negative for both the Flk-E1 and Flk-11 groups. Rank-log test comparing the Flk-E1 and Flk-11 groups to the control Lm groups give a highly significant value of *P < 0.0001.
Abbreviation: Lm, Listeria monocytogenes.
Table 1 Her-2/neu mutations in individual mice. All mutations that were found to repeat at least four times were selected as real induced mutations. Different sequencing reactions for each primer, forward and reverse, were used, and since multiple mRNA transcripts from multiple tumor cells were present in each tumor we find single amino acids undergoing several different mutations in a single mouse due to tumor heterogeneity. Control Lm, Flk-E1 and Flk-11 mice each had one tumor removed, mRNA extracted, cDNA transcribed, PCR amplified and sequenced using primers for overlapping regions of each of the five previously mapped Her-2/neu domains stretching 500–800 bp each (EC1 [AA 20–326], EC2 [AA 303–501], EC3 [AA 479–655], IC1 [AA 690–1081], IC2 [AA 1020–1260]). Bolded residues are those residues that undergo mutation when Her-2/neu directed vaccines were previously used to drive immunoediting of the Her-2/neu moleculeCitation11
Figure 2 Summary of individual mutations overlaid on the wild-type Her-2/neu amino acid sequence. Summary of all mutations were overlaid onto the full wild-type sequence of rat Her-2/neu. Bolded amino acids are residues that undergo mutation with the Her-2/neu Lm vaccine,Citation11 residues in dark shaded highlight are residues that undergo mutation after A) control B) Flk-E1, or C) Flk-11 Lm vaccination. Selected italic sequence denotes the kinase domain. Boxed sequences are previously identified CTL epitopes.Citation24 Sequence overlays contain compressed data from .
Abbreviation: CTL, cytotoxic T lymphocytes.
Figure 3 Mutated regions in Her-2/neu contain CTL epitopes. FVB/n wild-type mice were either immunized with Her-2/neu DNA vaccines or saline/PBS alone three sequential times each spaced one week. Splenocytes were tested ex vivo for their ability to recognize 20 mer peptides that overlapped mutated regions. Graph shows number of IFnγ spots per million splenocytes rechallenged using 20 mer peptides. Bolded residues are those amino acids that have undergone mutation using the Her-2/neu vaccines, italics are previously identified CTL epitopes,Citation24 and underlined residues are those amino acids that undergo mutation using Flk-1 vaccines. PMA/Ionomycin were used as a positive control (PosCtrol), pHIV Gag (AMQMLKETI) peptides was used as a third party control. Media alone well were subtracted from all groups as a background control. Graph shows mean ± SEM.
Abbreviations: CTL, cytotoxic T lymphocytes; PBS, phosphate-buffered saline; SEM, standard error of mean.
Figure 4 Epitope spreading to a previously unmapped Her-2/neu epitope occurs in response to FLK-1 vaccination in transgenic mice and T cell recognition is abrogated by accumulated mutations in this epitope. A) Wild-type and mutant peptides were synthesized for each region of interest from the neu1022–1041 20 mer peptide that showed the best response in re-challenge assays ex vivo and contained several mutations from both Her-2/neu and Flk-1 vaccinations. Mutations included a F1027I and g 1035K residue substitution. This substitution was based on prediction mapping using RANKpep and data from . B) Remaining mice that had not yet developed tumors, around week 55, were age-matched with saline-treated mice that had accumulated tumors and were analyzed for epitope spreading to this unmapped region and ability to recognize mutated versions that would most likely be expressed by the spontaneous tumors. Graph shows ex vivo IFNγ Elispot responses after rechallenge of splenocytes with the full 20 mer Neu1022–1041 peptide, and both fragments, Neu1026–1036 and Neu10321041, also mutant peptides NeuF1027I and neuG1035K pI1 = Flk-11906–915 (PGGPLMVIV), was used as a positive control for anti-Flk-11 responses. Media alone well were subtracted from all groups as a background control. Graph shows mean ± SEM; *P < 0.05; Mann–Whitney nonparametric test.
Abbreviation: SEM, standard error of mean.
