Immunization with an antigen identified by cytokine tumor vaccine-assistedSEREX (CAS) suppressed growth of the rat 9L glioma in vivo

We have reported previously that s.c. immunization of rats with IL-4 transd uced 9L gliosarcoma cells (9L-IL-4) induced a potent antitumor immunity aga inst intracranial, parental 9L tumors. Subcutaneous implantation of 9L-IL-4 influenced the systemic humoral response, which was demonstrated by Th2-ty pe isotype-switching and the induction of cellular immune responses, which played a critical role in the rejection of tumors. Serological analyses of recombinant cDNA expression libraries (SEREX), has recently emerged as a po werful method for serological identification of tumor-associated antigens ( TAAs) and/or tumor rejection antigens (TRAs). Because IL-4 is known to acti vate B cells and to promote humoral responses, and inasmuch as induction of humoral responses by central nervous system tumors has been reported to be minimal, me investigated whether the induction of a potent humoral immune response against 9L TAAs or TRAs in rats immunized s.c, with 9L-IL4 could b e demonstrated. Screening of 5 x 10(5) independent clones of 9L-expression cDNA library for the presence of reactive antibodies in the serum from a 9L -IL-4 immunized rat led to the identification of three different TAAs. One 9L TAA (clone 29) was demonstrated to be calcyclin, a member of the S-100 f amily of calcium-binding proteins. The second 9L TAA (clone 37) was demonst rated to be the rat homologue of the J6B7 mouse immunomodulatory molecule. The third TAA (clones 158 and 171) was determined to be the rat homologue o f the mouse Id-associated protein 1 (MIDA1), a DNA-binding, protein-associa ted protein. Northern blotting demonstrated that message for calcyclin was overexpressed in 9L cells. Message encoding (MIDA1 was highly expressed in parental 9L cells and thymus and, to a lesser degree, in testis, suggesting that MIDA1 was comparable with the cancer/testis category of TAAs. Sera ob tained from animals bearing 9L-IL-4 were found to have a higher a frequency and titer of antibodies to these antigens when compared with sera obtained from rats bearing sham-transduced 9L (9L-neo) cells. To determine whether immunization with these TAAs induced antitumor immunity, animals were immun ized by intradermal injection with expression plasmids encoding calcyclin o r MIDA1. Subsequent challenge of rats with parental 9L resulted in signific ant suppression of tumor growth in animals immunized with MIDA1, but not wi th calcyclin. These results indicate that MIDA1 is an effective 9L TRA and will be useful for the investigation of specific antitumor immunity in this glioma model. Furthermore, these results suggest that this approach, terme d "cytokine-assisted SEREX (CAS)," may serve as an effective strategy for i dentification of TRAs for in animal-glioma models of cytokine gene therapy, and potentially in humans undergoing cytokine gene therapy protocols as we ll.