Immunoproteomic Identification of Graft-Versus-Leukemia Targets in Patients with Chronic Lymphocytic Leukemia.

Chronic lymphocytic leukemia (CLL) is susceptible to immune-mediated destruction, as demonstrated by the potentially curative graft-versus-leukemia (GvL) effects of allogeneic hematopoietic stem cell transplant and donor lymphocyte infusion (DLI). Identification of the target antigens of GvL may lead to the development of novel reagents to monitor, diagnose or vaccinate against CLL. Our previous studies suggest that B cell responses are critical elements of successful GvL following DLI. In the current study, we used plasma from 2 patients who achieved durable complete remission after DLI for relapsed CLL as a source of immunoglobulin to probe high-density protein microarrays that express ∼5000 opening reading frames. As neither patient developed graft-versus-host disease (GvHD) after DLI, we hypothesized that immunologic dissection of responses in these patients would lead to the identification of CLL-associated antigens. Our studies identified 16 and 21 antigens with significantly higher reactivity post- versus pre-DLI for patients A and B, respectively. Specificity of candidate antigen-antibody interactions was validated by immunoprecipitation of individual recombinant proteins with patient plasma. Of 29 candidate antigen interactions tested, 21 (72%) were reactive, whereas all 5 control antigen interactions were unreactive. While most of the candidate antigens represent intracellular proteins of known function, nine are unknown. Of the known antigens, 8 bind nucleic acid and 2 are tumor suppressors, while others play roles in cellular functions such as ubiquitination, spermatogenesis, cell cycle, and apoptosis. Three proteins (C6orf130, MDS032, ZFYVE19) were recognized by both patients and therefore represent possible shared targets of anti-CLL immunity. By quantitative RT-PCR, all 3 shared antigens were more significantly expressed in malignant B cells than peripheral blood mononuclear cells, while one candidate, OGFOD1, was more significantly expressed in CLL than B cells, suggesting that immunogenicity may result from overexpression. Serologic characterization of reactivity patterns showed ZFYVE19 and DAPK3, another candidate, to be commonly immunogenic, as they elicited reactivity in 3 of 12 CLL patients in remission without GvHD post-transplant, in 0 of 12 normal volunteers and in only 1 of 12 untreated CLL patients. Confirming the close association of humoral immunity to GvL effects, histopathologic tumor regression was temporally related to presence of antibody reactivity against ZFYVE19 in both patients, to DAPK3 in patient A, and to OGFOD1 in patient B. Our results demonstrate that clinically evident anti-tumor immune responses in CLL are associated with the development of polyclonal humoral immunity, and that an immunoproteomic approach can rapidly identify CLL-associated antigens that are possible informative biomarkers of effective anti-CLL immunity. Ongoing studies will define the extent to which these targets elicit coordinated T cell mediated tumor rejection, and will elucidate their potential as novel immunogens for defined-antigen vaccines for CLL.