Patients with acute myeloid leukemia (AML) who have long-term disease-free survival following an allogeneic stem cell transplant (allo-SCT) may benefit from a robust immune response involving cytotoxic antibodies directed to a previously unsuspected cellular target, investigators from the Netherlands report.
The presence of these and other antibodies to newly identified cellular targets suggests new therapeutic strategies for treatment of AML and, potentially, for myelodysplastic syndromes (MDS), according to Mette D. Hazenberg, MD, PhD, from the Academic Medical Center in Amsterdam.
“What we learned from nature, from studying patients who through an allogeneic stem cell transplantation cured their leukemia, is that these patients make leukemia-specific antibodies to really unexpected targets,” she said in a report at the 2016 European Hematology Association (EHA) Congress in Copenhagen, Denmark.
By isolating and cloning anti-AML antibodies from long-term post-transplant survivors, Hazenberg and colleagues have identified antibodies that target small nuclear ribonucleoprotein U5 subunit 200 (U5 snRNP200). This large (250 kilodalton) protein is one of five major components of the spliceosome complex normally found within the nucleus of human and other eukaryotic cells, but which, as the team discovered, appears to be expressed on the cellular membrane of AML.
“It has been shown that allogeneic stem cell transplants can mount immune responses, and we wondered whether antibodies could be involved in this,” Hazenberg said.
She and her colleagues selected three patients with high-risk leukemia who were alive for at least 5 years with no evidence of disease following an allo-SCT, whose longevity indicated a strong graft-versus-leukemia response. The investigators identified and isolated antibody-generating memory B cells from peripheral blood and cultured them in medium, screening the supernatant of the cultures and searching for antibodies that bind to AML.
The team then cloned the AML-binding B cells and identified 17 candidate monoclonal antibodies that bind to AML in cell lines, but do not bind to peripheral blood monocytes, fibroblasts, or other healthy cells. Of the 17 antibodies, seven recognized the same target: U5 snRNP200.
“We screened a few more patients, and found that four out of five patients had mounted such antibodies, so apparently that is a target that is often recognized by the immune system of the donor,” Hazenberg said. “The second striking observation was that these antibodies, when they interact with this protein on the cell membrane, actually kill the leukemic cells.”
The direct killing effect was seen both in AML cells in vitro, and in a human AML mouse model in vivo. The cell death occurred despite the absence of either cytotoxic leukocytes or of complement. Instead, the antibodies appear to induce AML death through a non-apoptotic process that relies on destabilization of the cytoskeleton. The nature of the cell death mechanism was supported by further experiments showing that AML cell death could be blocked when target cells were treated with cytochalasin D, an inhibitor of actin polymerization.
Furthermore, observation that the anti-U5 snRNP200 antibodies retained their cytotoxic abilities at both 4° and 37° C suggests that the cell death is induced through a passive process. This observation was further supported by the fact that the interaction of the antibodies with their target cells did induce calcium flux, the investigators noted.
“And interestingly, when these antibodies bind to this complex they actually kill the target cell; they kill the leukemic cells. This is a novel phenomenon — we didn’t know this before — and we think we can develop this further into novel therapies.”
In a separate presentation at the EHA meeting, Hazenberg and colleagues reported on a second, novel tumor-specific target expressed on both AML and MDS blasts. The team first identified an immunoglobulin G1 antibody, labeled AT14-013, from the memory B lymphocytes of a patient with a robust graft-versus-leukemia response. This antibody homed in on a sialylated epitope of CD43 that is both uniquely and widely expressed on all types of AML.
Hazenberg and colleagues assert that antibodies targeted against onco-sialylated CD43 and U5 snRNP200 have significant potential as novel therapies for AML and MDS, either as “naked” antibodies or in combination in an antibody-drug conjugate, bispecific T-cell engager, or chimeric antigen receptor T cell (CAR-T) construct.