Background: Allogeneic hematopoietic stem cell transplantation (HSCT) can cure acute myeloid leukemia (AML) when the donor immune system generates a potent graft versus leukemia (GvL) response. While the role of T cells and NK cells in GvL immune responses has been established, the contribution of B cells to GvL responses is less clear. Using SEREX and other techniques, the presence of antibodies directed against established tumor antigens following allogeneic HSCT has been demonstrated, but because these antibodies were not obtained in monoclonal format, the function of these antibodies could not be analyzed.
Aim: To investigate the role of antibodies produced by donor-derived B cells in GvL responses.
Methods: We selected five patients with high-risk AML who remained disease-free for more than 5 years after allogeneic HSCT and thus have mounted a potent GvL response. From the peripheral blood of these patients we isolated memory B cells that we transduced with Bcl-6 and Bcl-xL, to establish antibody-producing clonal B cell lines. Blood was obtained 2 years after allogeneic HSCT. B cell lines were screened for the production of antibodies that specifically bound to surface antigens on AML cell lines and AML blasts isolated from patients in our clinic. Target identification was performed by immunoprecipitation and mass-spectometry.
Results: We identified patient derived clonal B cell lines producing antibodies that recognized antigens expressed on the cell surface of AML cells, but not on normal hematopoietic and non-hematopoietic cells. Antibodies were donor-derived, and a number of these antibodies recognized the U5 snRNP200 complex. The U5 snRNP200 complex is a component of the spliceosome that in normal cells is located in the nucleus but that is exposed on the cell membrane of AML cells. U5 snRNP200 complex-specific antibodies were specific for allogeneic HSCT recipients with AML, as they were found in in 4 out of 5 AML patients screened, but were not found in multiple myeloma patients who received an allogeneic HSCT or in healthy individuals. Strikingly, U5 snRNP200 complex-specific antibodies induced death of AML cells in vitro, and, in a human AML mouse model, in vivo. Cell death was induced in the absence of cytotoxic leukocytes or of complement, through a non-apoptotic process that depended on destabilization of the cytoskeleton as cell death could be blocked by incubation of the target cells with cytochalasin D, an actin polymerization inhibitor. Cytotoxicity of the U5 snRNP200 antibodies was present at 4°C and 37°C, suggesting that cell death was induced by a passive process. Indeed, interaction of the antibodies with their target cells did not induce a calcium flux. Cytotoxicity of the antibodies depended on the Fc region of the antibody, since recombinant U5 snRNP200 complex-specific antibodies with a defective Fc region were not cytotoxic.
Summary and Conclusions: Allogeneic HSCT recipients with robust donor anti-AML immunity generate antibodies against a component of the spliceosome, the U5 snRNP200 complex, that is expressed on the membrane of AML blasts. U5 snRNP200 antibodies are cytotoxic in vivo and in vitro, demonstrating the potency of the humoral immune system in tumor immunology.
Citation Format: Marijn Gillissen, Martijn Kedde, Greta De Jong, Etsuko Yasuda, Sophie Levie, Arjen Bakker, Yvonne Claassen, Koen Wagner, Julien Villaudy, Martino Bohne, Dave Speijer, Paul Hensbergen, Pauline van Helden, Tim Beaumont, Hergen Spits, Mette D. Hazenberg. Acute myeloid leukemia patients cured after allogeneic hematopoietic stem cell transplantation generate tumor-specific cytotoxic antibodies that kill AML blasts [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr A061.