Safety and Efficacy of Ex Vivo Expanded Healthy Donor-Derived Double Negative T Cells for the Treatment of AML Relapsed after Allogeneic Stem Cell Transplantation: A First in-Human Phase I/IIa Clinical Trial

Abstract

BACKGROUND Relapse after allogeneic hematopoietic stem cell transplantation (allo-HSCT) remains the main cause of treatment failure for patients with acute myeloid leukemia (AML). Conventional treatments such as donor lymphocyte infusions (DLI) or secondary allo-HSCT as a monotherapy or in combination with novel agents or immunotherapy showed limited improvements on long-term survival. Our previous studies have shown that a rare subset of CD4- and CD8- double negative T cells (DNTs) expanded from periphery of healthy donors are effective in targeting AML cells in vitro and in xenograft models. Also, allogeneic DNTs can be used as an off-the-shelf adoptive cellular therapy (ACT) for AML. We undertook a first in-human phase I/IIa trial to assess the safety and efficacy of escalating doses of DNTs for the treatment of AML patients relapsed post allo-HSCT. This study protocol was approved by Chinese Ethics Committee of Registering Clinical Trials. Informed consent was obtained in accordance with the Declaration of Helsinki. The trial was registered on the Chinese Clinical Trial Registry (ChiCTR-IPR-1900022795). METHODS From June 2019 to June 2020, 12 patients with relapsed AML after allo-HSCT were enrolled in the trials. Three patients received HLA-matched sibling stem cell transplant, and 9 patients received umbilical cord blood transplantation (UCBT). Hematological relapse was found in 9 cases, and molecular relapsed in 3 cases. None of the patients were able to obtain DLI from previous donors for various reasons. Clinical-grade DNTs from peripheral blood of healthy donors were expanded. After lymphodepleting chemotherapy with fludarabine and cyclophosphamide, each patient received three infusions of HLA-mismatched DNTs expanded from healthy volunteers at escalating doses of 5×107, 1×108, or 2-4×108 per kilogram of body weight at one week interval. Numbers of total DNTs and donor-derived DNTs, inflammatory cytokines levels in vivo were measured according to the study protocol. Post-remission therapy was permitted 14 days after the last DNTs infusion according to treating physician's discretion. RESULTS All surviving patients were followed until August 1st, 2020. At a median follow-up of 4.3 months (range 1.7 to 12.2 months), 1 patient withdrew from the trial after the second DNT infusion for a personal reason. 6/11 patients (54.5%) achieved complete response (CR) with 5 of these patients negative for (45.4%) minimal/measurable residual disease (MRD). 4/11 patients (36.3%) achieved partial remission. Up to the last follow-up, 4/11 patients remain CR. Increase in the levels of serum inflammatory cytokines, including interleukin (IL)-6, TNF-alpha, MCP-1, and MIP-1-beta over baseline were observed without signs of cytokine release syndrome, neurotoxicity, or graft-versus-host disease. The most common adverse events related to DNTs treatment were fever and headache. None of the patients showed >grade 2 adverse event (AE). The maximum tolerated dose was not reached. Donor DNT cell expansion was detected as early as 6 hours after infusion and reached the peak at 24 hours after the 1st infusion in most patients, slightly increased after the 2nd and 3rd infusions, and lasted for 2 weeks after 3rd infusion. However, total DNTs increased and reached the peak at 2 weeks after 3rd infusion. CONCLUSIONS Our results show that allogeneic DNTs therapy is safe, well tolerated, and potentially effective in treating AML patients who relapsed after allo-HSCT. The total number of DNTs increased after infusion. Compared to other agents, DNTs more effectively induced complete responses in this patient population. Our on-going study is to elucidate the mechanism of DNTs-mediated leukemia clearance, and explore the use of DNTs as carrier for Chimeric Antigen Receptor or transgenic TCR to further enhance the efficacy targeting resistant AML cells. Disclosures Zhang: AbbVie: Current equity holder in publicly-traded company; University Health Network: Consultancy, Current equity holder in private company, Other: Principal Investigator, Patents & Royalties.

Venetoclax enhances T cell–mediated antileukemic activity by increasing ROS production

“…However, we recognize that extrapolating the findings of DNTs to T conv cells should be done with caution. Because DNTs can be expanded ex vivo and have been safely administered to patients with relapsed and refractory AML, 28 there may be an opportunity to combine DNT therapy with venetoclax and azacytidine to further enhance their antileukemic activity.…”

Allogeneic double-negative CAR-T cells inhibit tumor growth without off-tumor toxicities

“…Furthermore, the risk of off-tumor toxicities can still persist with incomplete TCR knockout, as 10% of patients treated with allogeneic CAR-T convs knocked out for the endogenous TCR still developed GvHD in the clinic ( 10 ). A phase 1 clinical trial using NT-DNTs shows that none of the 12 AML patients treated with three doses of allogeneic NT-DNTs develop GvHD, grade >2 cytokine release syndrome (CRS), or neurotoxicity ( 25 ). Here, we showed that infusion of CAR19-DNTs without further genetic modification did not cause xenogeneic GvHD or any clinical symptoms of CRS or neurotoxicity.…”

“…Allogeneic DNTs also fulfill the requirements of an off-the-shelf adoptive cellular therapy, including scalability of expansion, donor-independent anticancer activity, cryopreservability, resistance to HvG rejection, and no observed off-tumor toxicity ( 18 ). A completed phase 1/2a clinical trial using third-party donor-derived genetically nonmodified DNTs to treat patients with relapsed/refractory acute myeloid leukemia (AML) shows that the therapy has a positive safety and efficacy profile ( 25 ).…”

CD3+CD4-CD8- (Double-Negative) T Cells in Inflammation, Immune Disorders and Cancer

“…Notably, none of the patients has developed GVHD or experienced adverse events higher than grade 2 thus far, while all patients experienced grade 1 or 2 cytokine release syndrome and then recovered quickly. Thus, this clinical study showed that allo-DN T cell therapy is safe and tolerable for AML patients, and an expanded patient cohort is urgently required for the next phase of the clinical trial ( 113 , 114 ).…”