Humoral response against a cell surface glycoantigen mediates apoptosis of tumor cells
Arkansas Cancer Research Center and Department of Pathology, University of Arkansas for Medical Sciences, Little Rock, AR, United States
Aim: Carbohydrate-reactive lectins have shown an ability to mediate apoptosis upon binding of tumor cells. Induction of humoral responses that mediate apoptosis is perceived to be beneficial in vaccine formulations. Here we show that immunization with a DNA form of a rationally chosen peptide mimotope of lectin-reactive tumor-associated carbohydrate antigen induces a humoral response that mediates apoptosis and significantly increases survival rate of tumor-bearing animal in the metastatic murine mammary 4T1 tumor model. Methods: Flow cytometry was utilized to profile 4T1 cell surface carbohydrates using plant lectins and for detection of apoptosis. An array of carbohydrate mimotopes was screened with the lectins by ELISA assay. Peptides were translated to DNA and cloned into a plasmid that is designed to secret the expressed product. Mice were inoculated with 4T1 cells in the abdominal mammary gland. DNA immunization started 4 days post-tumor transplant and each mouse received three intramuscular injection of each construct. Tumor size was measured regularly and survival date recorded. Lectin histochemistry was performed using organs harvested from tumor-bearing mice. Metastasis was assessed by growth in selective medium. Results: We observed that Griffonia simplicifolia lectin I (GS-I) binds to 4T1 cells. A peptide mimotope that was chosen based on its reactivity with GS-I inhibits the binding of the lectin to 4T1 cells, indicating that the peptide mimic’s a carbohydrate structure reactive with GS-I. By analysis of annexin V binding we observed that GS-I induces apoptosis in 4T1 cells. Immunization with the peptide mimic generates antibodies that bind to the cells and induce apoptosis of the tumor cells. We further observed that therapeutic immunization of tumor-bearing animals with the peptide-encoding construct results in slight tumor shrinkage but statistically significant (P = 0.021) higher survival rate as compared with vector immunized animals. Immunization with the peptide significantly diminished metastasis to the liver (P= 0.018). Histochemistry demonstrates that all tumor cells in liver are stained with GS-I, while staining is partial for tumor cells in the primary mass and lung. The Histochemical data imply that liver metastasis might depend on the presence of GS-I-reactive structure on migrating cells. Conclusions: Here we report that a peptide surrogate of a tumor associated carbohydrate antigen can trigger an in vivo tumor-specific response in which serum antibodies function as mediators of cell apoptosis. Induction of apoptosis in combination with activation of carbohydrate targeting cellular immune responses might be an effective strategy for vaccination against weakly immunogenic malignancies.
Paper presented at the International Symposium on Predictive Oncology and Intervention Strategies; Nice, France; February 7 - 10, 2004; in oral session 995 (Immunotherapy).