Effect of a novel DNA vaccine on angiogenesis and tumor growth in vivo.

OBJECTIVE: To develop a DNA cancer vaccine that targets the vascular endothelial growth factor receptor. DESIGN: Mice were vaccinated intramuscularly with listeriolysin O-fetal liver kinase 1 (LLO-Flk1) or controls. Mice were also challenged subcutaneously with the tumor cell line TC-1. Tumor sizes were measured after vaccination. At the conclusion of the experiments, the tumors were harvested for immunohistochemical analysis and determination of hemoglobin content. SETTING: Research laboratory. SUBJECTS: Six- to 8-week-old C57BL/6 mice. INTERVENTION: Fifty micrograms of each vector was administered 3 times at weekly intervals. MAIN OUTCOME MEASURES: Tumor size, mean vessel density of tumors, hemoglobin content of tumor. RESULTS: Mice treated with the LLO-Flk1 vaccine experienced slower tumor growth relative to the other treatment groups. Complete tumor regression was observed in several cases. Immunohistochemical staining of tumors revealed fewer blood vessels in the mice vaccinated with the LLO-Flk1 vaccine relative to the other treatment groups. Finally, colorimetric assessment for hemoglobin suggested decreased vasculature in the tumor bed of these mice relative to the control groups. CONCLUSION: The novel DNA cancer vaccine LLO-Flk1 can slow tumor growth in vivo.

Deletion of Candida albicans SPT6 is not lethal but results in defective hyphal growth.

As a means to study surface proteins involved in the yeast to hypha transition, human monoclonal antibody fragments (single-chain variable fragments, scFv) have been generated that bind to antigens expressed on the surface of Candida albicans yeast and/or hyphae. A cDNA expression library was constructed from hyphae, and screened for immunoreactivity with scFv5 as a means to identify its cognate antigen. A reactive clone contained the 3' end of the C. albicans gene, orf 19.7136, designated SPT6 based on homology to Saccharomyces cerevisiae, where its product functions as a transcription elongation factor. A mutant containing a homozygous deletion of SPT6 was isolated, demonstrating that unlike S. cerevisiae, deletion of this gene in C. albicans is not lethal. Growth of this strain was severely impaired, however, as was its capacity to undergo filamentous growth. Reactivity with scFv5 was not detected in the mutant strain, although its impaired growth complicates the interpretation of this finding. To assess C. albicansSPT6 function, expression of the C. albicans gene was induced in a defined S. cerevisiaespt6 mutant. Partial complementation was seen, confirming that the C. albicans and S. cerevisiae genes are functionally related in these species.

An anti-vascular endothelial growth factor receptor 2/fetal liver kinase-1 Listeria monocytogenes anti-angiogenesis cancer vaccine for the treatment of primary and metastatic Her-2/neu+ breast tumors in a mouse model.

Thirty years after angiogenesis was shown to play an enabling role in cancer, modern medicine is still trying to develop novel compounds and therapeutics to target the tumor vasculature. However, most therapeutics require multiple rounds of administration and can have toxic side effects. In this study, we use anti-angiogenesis immunotherapy to target cells actively involved in forming new blood vessels that support the growth and spread of breast cancer. Targeting a central cell type involved in angiogenesis, endothelial cells, we immunized against host vascular endothelial growth factor receptor 2 to fight the growth of Her-2/neu(+) breast tumors. Using the bacterial vector, Listeria monocytogenes (Lm), we fused polypeptides from the mouse vascular endothelial growth factor receptor 2 molecule (fetal liver kinase-1) to the microbial adjuvant, listeriolysin-O, and used Lm to deliver the Ags and elicit potent antitumor CTL responses. Lm-listeriolysin-O-fetal liver kinase-1 was able to eradicate some established breast tumors, reduce microvascular density in the remaining tumors, protect against tumor rechallenge and experimental metastases, and induce epitope spreading to various regions of the tumor-associated Ag Her-2/neu. Tumor eradication was found to be dependent on epitope spreading to HER-2/neu and was not solely due to the reduction of tumor vasculature. However, vaccine efficacy did not affect normal wound healing nor have toxic side effects on pregnancy. We show that an anti-angiogenesis vaccine can overcome tolerance to the host vasculature driving epitope spreading to an endogenous tumor protein and drive active tumor regression.