A novel CD34-specific T-cell engager efficiently depletes acute myeloid leukemia and leukemic stem cells <i>in vitro</i> and <i>in vivo</i>

Abstract

Less than a third of acute myeloid leukemia (AML) patients are cured by chemotherapy and/or hematopoietic stem cell transplantation (HSCT), highlighting the need to develop more efficient drugs. The low efficacy of standard treatments is associated with inadequate depletion of CD34+ blasts and leukemic stem cells (LSCs), the latter a drug-resistant subpopulation of leukemia cells characterized by the CD34+CD38- phenotype. To better target these drug-resistant primitive leukemic cells, we have designed a CD34/CD3 bispecific T-cell engager (BiTE) and characterized its anti-leukemia potential in vitro, ex vivo and in vivo. Our results show that this CD34-specific BiTE induces CD34-dependent T-cell activation and subsequent leukemia cell killing in a dose-dependent manner, further corroborated by enhanced T-cell-mediated killing at the single-cell level. Additionally, the BiTE triggered efficient T-cell-mediated depletion of CD34+ HSCs from peripheral blood stem cell grafts and CD34+ blasts from AML patients. Using a humanized AML xenograft model, we confirmed that the CD34-specific BiTE had in vivo efficacy by depleting CD34+ blasts and LSCs without side effects. Taken together, these data demonstrate robust antitumor effects of the CD34-specific BiTE supporting development of a novel treatment modality with the aim of improving outcomes of patients with AML and myelodysplastic syndromes.

Selective lysis of acute myeloid leukemia cells by CD34/CD3 bispecific antibody through the activation of γδ T-cells

“… 39–45 We have developed a CD34-specific BTE with the aim of eradicating CD34+ HSCs and AML blasts (including LSCs) prior to HSCT. 23 The CD34-specific BTE could, theoretically, be used in combination with chemotherapy as an adjuvant therapy before transplantation to lower LSC burden and potentially reduce the risk of an LSC-driven relapse post-HSCT. Moreover, the CD34-specific BTE may also allow for a lower dosage of chemotherapeutics to be used before transplantation in hopes of minimizing drug-related toxicities.…”

“…Target cell killing by effector T-cells was performed as described previously. 23 In short, effector and target cells (cancer cell lines, primary AML, or healthy BM) were thawed, and allowed to rest overnight at 37 °C in complete culture medium. The following day, effector γδ T-cells, αβ T-cells, and αβ T-cells pre-stimulated with OKT3, were co-cultured with target cells at a 3:1 ratio with serial dilutions of BTE (0.1, 1, 10, 100, and 1000 ng/mL) for 4 h, 24 h, 48 h, 72 h, and 120 h. Target cells (4 h, 24 h, 48 h and 72 h assays) or effector cells (120 h assay) were pre-stained with 2 uM of CellTrace Violet (Thermo Fisher), according to manufacturer’s instructions, to help differentiate between target and effector cells by flow cytometry.…”

“…The proliferation capacity of expanded effector γδ T-cells in response to exposure to target cancer cell lines and BTE was assessed as described previously. 23 In short, effector γδ T-cells were pre-stained with 2 uM of CellTrace Violet and incubated for 5 d with target cells (SUPB15, Kasumi, KG1a, and NALM-6) at a 3:1 ratio with serial dilutions of BTE (0.1, 1, 10, 100, and 1000 ng/mL). The fraction of CellTracelow proliferating γδ T-cells was measured by flow cytometry.…”

“…The binding efficacy of BTE to γδ T-cells and cancer cell lines was measured as previously described. 23 In short, cells were incubated with BTE at a concentration of 1000 ng/mL for 20 min at 4 °C. After incubation, cells were washed with PBS, and normal extracellular staining was performed with a secondary anti-His tag antibody and relevant antibodies as described in the flow cytometry section.…”

“…In our pre-clinical investigation of a novel CD34-specific BTE, we demonstrated its ability to selectively lyse AML cell lines and leukemic blasts in vitro and reduce tumor burden in vivo . 23 Moreover, CD34 intermediately positive endothelial cell lines were not killed, suggesting its non-toxicity toward these cells. In our previous investigation, BTEs were tested with conventional T-cells.…”