Abstract 2478: A mAb with in vivo therapeutic activity against spontaneous breast tumors in FVB-neuN mice recognizes SLP-2

BCSC (breast cancer stem cells) derived from FVB-neuN mice bearing primary breast tumors were used to vaccinate mice and vaccine-draining lymph node T cells were adoptively transferred to mice with advanced primary tumors. Treated mice experienced tumor regression and importantly a concomitant serologic response was induced to an antigen that is expressed by BCSC even in Neu-antigen loss tumor cells. We generated a specific mAb (mAb2) reactive against a membrane antigen on BCSC tumorsphere cultures derived from NeuN spontaneous breast tumors but not the counterpart adherent cultures. In vivo administration of mAb2 inhibited progression of macroscopic primary tumors in Her-2/neu transgenic mice. We further observed that the molecule bound by mAb2 is expressed on proliferating but not growth-arrested C2D1-tumorspheres and this antigen is re-expressed when cells are digested into single cell suspension and re-fed with fresh medium. Similar results are observed with 4T1-tumorsphere cells, an additional breast cancer cell line arising initially in BALB/c mice. Interestingly, the target of mAb2 is also down-regulated following irradiation-induced growth arrest of tumor cells. To identify the antigen recognized by mAb2, cultured C2D1-tumorsphere or 4T1-tumorspheres were cells were lysed and the supernatant was immunoprecipitated with mAb2. The proteins captured by mAb2 were separated by 10% SDS-PAGE and visualized by silver staining. The bands of interest were excised then subjected to liquid chromatography-mass spectrometric (LC-MS/MS) analysis. A ∼40 KDa protein was detected in all five experiments from either C2D1-tumorsphere or 4T1-tumorsphere cells. Stomatin-like protein 2 (SLP-2) a 38.5 KDa protein was consistently identified whereas 14 other proteins were sporadically found. Western blots of mAb2 immunoprecipitates from C2D1 or 4T1-tumorsphere lysates were probed with two distinct commercial antibodies against SLP-2 and we confirmed its presence. To validate SLP-2 as the target, an expression vector was transfected into NIH 3T3 cells and FACS analysis demonstrated 19% positive cells versus 3.6% for un-transfected 3T3 cells. SLP-2 overexpression is associated with tumor metastasis and poor prognosis in several types of human tumors. Our results indicate that the antigen recognized by this auto-antibody might have immunodiagnostic or immunotherapeutic potential. Citation Format: Li-Xin Wang, Michael Berk, Gregory E. Plautz. A mAb with in vivo therapeutic act