AbstractAbstract 463We recently reported that vaccination with autologous monocyte-derived dendritic cells pulsed with dying autologous tumor cells elicited a clinical response strongly associated with multifaceted antitumor immune-activation in relapsed indolent non-Hodgkin lymphoma (NHL) patients. We have now set out to determine whether vaccine-induced humoral response is directed against common indolent NHL-restricted antigens (ags), which could thus be exploited as novel targets for therapy. Antibodies (Abs) were purified from pre- and post-vaccine patients' serum samples, biotin-conjugated, and initially tested by immunohistochemistry (IHC) and flow cytometry (FC) on allogeneic tumors biopsies or live tumor cells, both primary tumors and cell lines. We found that post-vaccine Abs from responders (R) reacted with allogeneic NHL at significantly higher levels than their matched pre-vaccine samples or non-R Abs. Furthermore, Rs' post-vaccine sera significantly impaired the growth of DOHH-2 and RL-19 follicular lymphoma (FL) cell lines, as revealed by standard 3-(4,5- dimethylthiazol- 2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays. To identify the therapeutically targeted NHL ags, we then used biotin-conjugated patient Abs to immunoblot one-dimensional SDS-PAGE of DOHH-2 cell protein fractions obtained by isoelectrofocusing. Protein bands differentially revealed by post-vaccine Abs from R were analyzed by Mass Spectrometry (MS). One differential band migrating at about 100 kDa was revealed among the most acidic protein fractions only when post-vaccine samples from R were used. MS analysis identified heat shock protein (HSP) 105 in the differentially reacting bands. Immunoprecipitation with a commercial anti-HSP105 Ab followed by Western blot analysis with biotin-conjugated pre- and post-vaccine Abs from R confirmed the increased ability of the post-vaccine sample to recognize HSP105. FC disclosed HSP105 both on the cell membrane and in the cytoplasm of a panel of B-cell NHL cell lines and normal B cells. The extent of HSP105 surface expression increased in function of lymphoma istotype aggressiveness. The antitumor activity of anti-HSP105 Ab, measured by MTT assays, was thus higher against Burkitt's lymphoma (BL) cell lines than germinal centre-derived diffuse large B cell lymphoma and FL cell lines, which displayed an IC50 of 4.5, 7.5, 11.7 μg/ml, respectively. To confirm these finding in primary human tumors, we performed IHC analyses of HSP105 on 68 diagnostic NHL specimens (35 low-grade and 33 high-grade NHL) and 23 non-malignant lymph nodes obtained from our Institutional Tissue Bank. Low-grade NHL's cytoplasmic immunoreactivity was mainly restricted to actively proliferating cells (i.e. Ki67 positive germinal centre cells), whereas high-grade NHL displayed a significantly higher expression of HSP105, measured both as intensity and percentage of positive cells (p=0.0002). In addition, malignant cells in high-grade NHL more often displayed a specific cell-surface staining. Interestingly, the expression pattern and intensity of HSP105 was widely superimposable on that of the proliferation marker Ki67, as detected by the specific monoclonal Ab Mib-1. Lastly, the therapeutic effects of HSP105 functional inhibition was studied in Namalwa BL xenotransplanted SCID mice using a specific commercial Ab. Treated mice showed a significant delay in tumor growth compared to untreated control animals (p=0.0014). Taken as a whole, our results indicate that HSP105 could be a new potential biotarget for the treatment of NHL and a novel candidate biomarker for an improved management of B-cell lymphoma. Its location on the normal B cell surface, and its increasing expression with NHL aggressiveness open a new area in which to assess its role in B-cell biology and lymphoma physiopathology. Disclosures:No relevant conflicts of interest to declare.