Identification by Serological Proteome Analysis (SERPA) of Tumor-Associated Antigens Eliciting Antibody Responses In Chronic Lymphocytic Leukemia (CLL)
Authors: Coscia Marta , Capello Michela , Griggio Valentina , Linty Federica , Vitale Candida , Peola Silvia , Castella Barbara , Rigoni Micol , Drandi Daniela , Ruggeri Marina , Omedè Paola , Ladetto Marco , Novelli Franco , Boccadoro Mario , Massaia Massimo 
Published: October 2019

Abstract
Abstract 917
In this study we applied the serological proteomics-based approach (SERPA) to identify novel tumor associated antigens (TAA) capable of inducing humoral immune responses in patients with chronic lymphocytic leukemia (CLL). Proteins extracted from the leukemic cells isolated from the peripheral blood of 21 untreated CLL patients were separated by 2-DE electrophoresis and transferred onto membranes by electroblotting to obtain 21 2-DE proteomic maps. Each map was subsequently probed with the corresponding autologous serum collected from the same patient. To verify the CLL-specificity of antibodies (Ab) recognition, 7 out of 21 maps obtained from CLL patients were also probed with sera collected from 7 healthy donors (HD). The Western Blot (WB) performed with sera of CLL patients displayed a total of 45 immunoreactive spots. Only 3 antigen spots were detected in HD sera. For identification, antigen spots in WB were aligned with proteins in 2-DE. The protein spots corresponding to the assigned antigens were excised from the gel, destained and subjected to trypsin digestion. The resulting tryptic fragments were analyzed by peptide mass fingerprint by MALDITOF-MS with MASCOT. All the 45 antigen spots were characterized and consisted of 16 different antigens. Sixteen out of 21 CLL sera (76%) showed immunoreactivity against at least 1 of the 16 identified TAA and 69% of these reactive sera recognized from 2 to 6 different antigens. The IGHV mutational status was available in 20 CLL patients and 12 patients were M, while 8 patients were UM. The reactivity rate and number of WB spots were similar in M and UM patients and did not correlate with other parameters of clinical outcome.
Sera from 46% CLL patients exhibited immunoreactivity against a protein which was identified by mass spectrometry as α-Enolase (ENOA). Interestingly, ENOA recognition was CLL specific since none of the sera from HD showed reactivity against this protein. The frequency of ENOA recognition was particularly high in M patients. Indeed, ENOA was recognized from sera of 7 out of 12 M patients (59%), but only from sera of 2 out of 8 UM patients (25%).
The ability of ENOA to induce antigen-specific T cell responses was assessed. T cells isolated from the PB of a CLL patient with Ab-based ENOA reactivity were stimulated with autologous monocytes-derived ENOA-pulsed dendritic cells (DC). The results showed that CLL-derived ENOA-pulsed DC stimulated autologous T cells to secrete IFN-gamma. This response was ENOA-specific because it was not induced by unpulsed DC or DC pulsed with an irrelevant protein, and also CLL-specific because IFN-gamma release was not induced when T cells from a HD were stimulated with autologous ENOA-pulsed DC.
Altogether, these results indicate that ENOA is capable of eliciting CLL-specific humoral and cellular immune responses. Therefore, ENOA can be considered as an alternative and promising biomarker in CLL, as well as a potential target candidate for immunotherapeutic approaches.

Disclosures:
Boccadoro: Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen-Cilag: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding. Massaia:Novartis: Honoraria, Research Funding.