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Acute myeloid leukemia (AML) is an immune-susceptible malignancy, as demonstrated by its responsiveness to allogeneic stem cell transplantation (alloSCT). However, by employing inhibitory signaling pathways, including PD-1/PD-L1, leukemia cells suppress T cell-mediated immune attack. Notably, impressive clinical efficacy has been obtained with PD-1/PD-L1 blocking antibodies in cancer patients. Yet, these systemic treatments are often accompanied by severe toxicity, especially after alloSCT. Here, we investigated RNA interference technology as an alternative strategy to locally interfere with PD-1/PD-L1 signaling in AML. We demonstrated efficient siRNA-mediated PD-L1 silencing in HL-60 and patients’ AML cells. Importantly, WT1-antigen T cell receptor+PD-1+2D3 cells showed increased activation toward PD-L1 silenced WT1+AML. Moreover, PD-L1 silenced AML cells significantly enhanced the activation, degranulation, and IFN-? production of minor histocompatibility antigen-specific CD8+T cells. Notably, PD-L1 silencing was equally effective as PD-1 antibody blockade. Together, our study demonstrates that PD-L1 silencing may be an effective strategy to augment AML immune-susceptibility. This provides rationale for further development of targeted approaches to locally interfere with immune escape mechanisms in AML, thereby minimizing severe toxicity. In combination with alloSCT and/or adoptive T cell transfer, this strategy could be very appealing to boost graft-versus-leukemia immunity and improve outcome in AML patients.

Effective T-cell therapy against cancer is dependent on the formation of long-lived, stem cell–like T cells with the ability to self-renew and differentiate into potent effector cells. Here, we investigated the in vivo existence of stem cell–like antigen-specific T cells in allogeneic stem cell transplantation (allo-SCT) patients and their ex vivo generation for additive treatment posttransplant. Early after allo-SCT, CD8+ stem cell memory T cells targeting minor histocompatibility antigens (MiHAs) expressed by recipient tumor cells were not detectable, emphasizing the need for improved additive MiHA-specific T-cell therapy. Importantly, MiHA-specific CD8+ T cells with an early CCR7+CD62L+CD45RO+CD27+CD28+CD95+ memory-like phenotype and gene signature could be expanded from naive precursors by inhibiting Akt signaling during ex vivo priming and expansion. This resulted in a MiHA-specific CD8+ T-cell population containing a high proportion of stem cell–like T cells compared with terminal differentiated effector T cells in control cultures. Importantly, these Akt-inhibited MiHA-specific CD8+ T cells showed a superior expansion capacity in vitro and in immunodeficient mice and induced a superior antitumor effect in intrafemural multiple myeloma–bearing mice. These findings provide a rationale for clinical exploitation of ex vivo–generated Akt-inhibited MiHA-specific CD8+ T cells in additive immunotherapy to prevent or treat relapse in allo-SCT patients.

Effective T-cell therapy against cancer is dependent on the formation of long-lived, stem cell–like T cells with the ability to self-renew and differentiate into potent effector cells. Here, we investigated the in vivo existence of stem cell–like antigen-specific T cells in allogeneic stem cell transplantation (allo-SCT) patients and their ex vivo generation for additive treatment posttransplant. Early after allo-SCT, CD8+stem cell memory T cells targeting minor histocompatibility antigens (MiHAs) expressed by recipient tumor cells were not detectable, emphasizing the need for improved additive MiHA-specific T-cell therapy. Importantly, MiHA-specific CD8+T cells with an early CCR7+CD62L+CD45RO+CD27+CD28+CD95+memory-like phenotype and gene signature could be expanded from naive precursors by inhibiting Akt signaling during ex vivo priming and expansion. This resulted in a MiHA-specific CD8+T-cell population containing a high proportion of stem cell–like T cells compared with terminal differentiated effector T cells in control cultures. Importantly, these Akt-inhibited MiHA-specific CD8+T cells showed a superior expansion capacity in vitro and in immunodeficient mice and induced a superior antitumor effect in intrafemural multiple myeloma–bearing mice. These findings provide a rationale for clinical exploitation of ex vivo–generated Akt-inhibited MiHA-specific CD8+T cells in additive immunotherapy to prevent or treat relapse in allo-SCT patients.

ABSTRACTCombining natural killer (NK) cell adoptive transfer with tumor-sensitizing chemotherapy is an attractive approach against recurrent ovarian cancer (OC), as OC is sensitive to NK cell-mediated immunity. Previously, we showed that CD34+hematopoietic progenitor cell (HPC)-derived NK cells can kill OC cells in vitroand inhibit OC tumor growth in mice. Here, we investigated the potential of HPC-NK cell therapy combined with chemotherapeutic gemcitabine (used in recurrent OC patients) against OC. We examined the phenotypical, functional, and cytotoxic effects of gemcitabine on HPC-NK cells and/or OC cells in vitroand in OC-bearing mice. To this end, we treated OC cells and/or HPC-NK cells with or without gemcitabine and analyzed the phenotype, cytokine production, and anti-tumor reactivity. We found that gemcitabine did not affect the phenotype and functionality of HPC-NK cells, while on OC cells expression of NK cell activating ligands and death receptors was upregulated. Although gemcitabine pre-treatment of OC cells did not improve the functionality of HPC-NK cells, importantly, HPC-NK cells and gemcitabine additively killed OC cells in vitro. Similarly, combined HPC-NK cell and gemcitabine treatment additively decreased tumor growth in OC-bearing mice. Collectively, our results indicate that combination therapy of HPC-NK cells and gemcitabine results in augmented OC killing in vitroand in vivo. This provides a rationale for exploring this therapeutic strategy in patients with recurrent OC.

ABSTRACTAdoptive T cell therapy has shown clinical potential for patients with cancer, though effective treatment is dependent on longevity and potency of the exploited tumor-reactive T cells. Previously, we showed that ex vivoinhibition of AKT using the research compound Akt-inhibitor VIII retained differentiation and improved functionality of minor histocompatibility antigen (MiHA)-specific CD8+T cells. Here, we compared a panel of clinically applicable AKT-inhibitors with an allosteric or adenosine triphosphate-competitive mode of action. We analyzed phenotype, functionality, metabolism and transcriptome of AKT-inhibited CD8+T cells using different T cell activation models. Most inhibitors facilitated T cell expansion while preserving an early memory phenotype, reflected by maintenance of CD62L, CCR7 and CXCR4 expression. Moreover, transcriptome profiling revealed that AKT-inhibited CD8+T cells clustered closely to naturally occurring stem cell-memory CD8+T cells, while control T cells resembled effector-memory T cells. Interestingly, AKT-inhibited CD8+T cells showed enrichment of hypoxia-associated genes, which was consistent with enhanced glycolytic function. Notably, AKT-inhibition during MiHA-specific CD8+T cell priming uncoupled preservation of early memory differentiation from ex vivoexpansion. Furthermore, AKT-inhibited MiHA-specific CD8+T cells showed increased polyfunctionality with co-secretion of IFN-γ and IL-2 upon antigen recall. Together, these data demonstrate that AKT-inhibitors with different modality of action promote the ex vivogeneration of stem cell memory-like CD8+T cells with a unique metabolic profile and retained polyfunctionality. Akt-inhibitor VIII and GDC-0068 outperformed other inhibitors, and are therefore promising candidates for ex vivogeneration of superior tumor-reactive T cells for adoptive immunotherapy in cancer patients.

Spanholtz: Glycostem Therapeutics: Employment. Tordoir:Glycostem Therapeutics: Employment. Bohme:Glycostem Therapeutics: Employment. Kok:Glycostem Therapeutics: Employment.

Introduction

Adoptive transfer of allogeneic natural killer (NK) cells represents a promising treatment approach against cancer, including acute myeloid leukemia (AML). Previously, we reported a cytokine-based culture method for the generation of NK cell products with high cell number and purity. In this system, CD34+hematopoietic progenitor cells (HPC) were expanded and differentiated into NK cells under stroma-free conditions in the presence of IL-15 and IL-2. We show that combining IL-15 with IL-12 drives the generation of more mature and highly functional NK cells. In particular, replacement of IL-2 by IL-12 enhanced the cytolytic activity and IFNγ production of HPC-NK cells toward cultured and primary AML cells in vitro, and improved antileukemic responses in NOD/SCID-IL2Rγnull (NSG) mice bearing human AML cells. Phenotypically, IL-12 increased the frequency of HPC-NK cells expressing NKG2A and killer immunoglobulin-like receptor (KIR), which were more responsive to target cell stimulation. In addition, NK15/12 cell products demonstrated superior maturation potential, resulting in >70% positivity for CD16 and/or KIR within 2 weeks after infusion into NSG mice. We predict that higher functionality and faster in vivomaturation will favor HPC-NK cell alloreactivity toward malignant cells in patients, making this cytokine combination an attractive strategy to generate clinical HPC-NK cell products for cancer adoptive immunotherapy.

T cells are crucial players in the protection against cancer, and can be used in adoptive cell therapy to prevent or treat relapse. However, their state of differentiation determines their effectiveness, with early memory cells being the most favorable. Here, we discuss restraining of differentiation to engineer the ultimate tumor-reactive T cell.

No relevant conflicts of interest to declare.

Natural killer (NK) cell-based immunotherapy is a promising adjuvant, relatively non-toxic therapy approach for AML. However, further improvement of NK cell-based therapy is needed to increase the clinical effect. In this regard, NK cells generated ex vivofrom hematopoietic progenitor cells (HPC) may have significant clinical benefits over enriched NK cells from adult donors, including the ability to choose an appropriate killer-cell immunoglobuline-like receptor (KIR)-ligand or KIR B haplotype alloreactive donor, as well as the capacity to reach high therapeutic dosages. Previously, we reported a GMP-compliant, cytokine/heparin-based culture protocol for the ex vivogeneration of highly active NK cells from CD34+HPC isolated from cryopreserved umbilical cord blood (UCB) units. Expansion in closed, large-scale bioreactors yields a clinically relevant dose of NK cells with high purity and cytolytic activity against AML cells in vitro.Currently, a clinical phase I trial with these HPC-NK cells is ongoing in our hospital. Trafficking studies in NOD/SCID/IL2Rgnull(NSG) mice demonstrated that these HPC-NK cells migrate to the bone marrow (BM) as well as to lymphoid organs where in vivoexpansion and maturation can take place. Analysis of the chemokine receptor expression profile of UCB-NK cells matched in vivofindings. Particularly, a firm proportion of UCB-NK cells functionally expressed CXCR4, what could trigger BM homing in response to its ligand CXCL12. In addition, high expression of CXCR3 and CCR6 supported the capacity of UCB-NK cells to migrate to inflamed tissues via the CXCR3/CXCL10-11 and CCR6/CCL20 axis. Importantly, a single HPC-NK cell infusion combined with supportive IL-15 administration was shown to efficiently inhibit growth of K562 leukemia cells implanted in the femur of NSG mice, resulting in significant prolongation of mice survival. Furthermore, we investigated whether modulation by the DNA methyltransferase (DNMT) inhibitors Azacytidine (Aza) and Decitabine (Deci) could further potentiate the antileukemic effect of HPC-NK cells against AML cells. In concordance with previous reports, we observed a dose-dependent effect of Aza and Deci on the growth of the AML cell lines THP1 and KG1a. In subsequent NK cell killing assays, we used clinical relevant low drug concentrations to pre-treat AML cells that did not affect HPC-NK cell viability and cytolytic function. Interestingly, increased killing of pre-treated THP1 and KG1a cells by HPC-NK cells could be observed, which was correlated with an increase in the NKG2D ligand ULBP2, the DNAM-1 ligands CD112 and CD155 as well as TRAIL-R2. Notably, maintenance of low-dose DNMT inhibitors during the KG1a/NK co-culture resulted in pronounced AML growth inhibition. To examine the effect of DNMT inhibitors in vivo, THP1.LucGFP-bearing NSG mice were treated with increasing dose of both agents, which were administered according to current standard protocols applied in humans. Data indicated that treatment with Aza or Deci at dosage equivalent in human to 12.5 and 5 mg/m2respectively was well tolerated with minimal and/or transient weight loss, and efficiently reduced the progression of THP-1.LucGFP cells in vivo. Currently, we explore whether HPC-NK cells and DNMT inhibitors can work together to combat AML in our xenograft models. These preclinical studies may provide a rationale to investigate the possible additive and/or synergistic anti-AML effects of adoptive HPC-NK cell transfer in combination with these DNMT inhibitors in AML patients.

Allogeneic hematopoietic stem cell transplantation (allo-SCT) followed by donor lymphocyte infusion (DLI) is a potential curative treatment for patients suffering from a hematological malignancy. Efficacy is attributed to the graft-versus-tumor (GVT) response, during which engrafted donor T cells become activated by recipient minor histocompatibility antigens (MiHA) presented on dendritic cells (DC). Subsequently, these activated T cells expand, acquire effector functions and kill MiHA-positive tumor cells. However, persistence and recurrence of malignant disease is often observed, indicating that insufficient GVT immunity is induced. This imperfect alloreactive response is probably due to insufficient numbers of MiHA-specific effector T cells and/or defective antigen-presentation and costimulation. Therefore, adoptive transfer of potent ex vivo-generated MiHA-specific T cells, restricted to the hematopoietic system, would boost the GVT-effect without increasing the risk for GVHD. Although successful in vitroinduction of MiHA-specific CD8+ T cells from naive precursors has been reported, the resulting antigen-experienced T cell population consist of fully differentiated effector-memory T cells (TEM). Over the past years it has been described that this T cell subset is not the most potent memory subset in anti-tumor responses in vivofollowing T cell transfer. In this regard, the less-differentiated memory subset called stem cell memory T cells (TSCM) with superior in vivoexpansion, self-renewal capacity and plasticity to differentiate in potent effectors would generate a stronger GVT response. In this study, we aimed to investigate the in vivoavailability and ex vivogeneration of TSCM-like MiHA-specific T cells as additive treatment option for allo-SCT patients. First, we investigated whether in allo-SCT patients MiHA-specific T cells could be detected with a TSCMphenotype defined by the expression of CD45RO, CCR7, CD27 and CD95. Though TSCMcells could be clearly detected within CMV-specific CD8+ T cells in allo-SCT patients, similar to healthy controls, no MiHA-specific TSCMcells could be detected. This emphasises the need for more potent adoptive MiHA-specific T cell therapy following allo-SCT. Therefore, we next explored the possibility of generating TSCM-like CD8+ T cells by interfering with the Akt signalling pathway. Emerging findings indicate that the differentiation program of CD8+ T cells is dictated by the strength and duration of AKT activity. Therefore, we explored whether the pharmacological inhibition of this signaling pathway could results in the generation of TSCM-like CD8+ T cells. We stimulated CCR7+CD45RA+ naive CD8+ T cells with CD3/CD28 beads plus IL-2, IL-7 and/or IL-15 in the presence an Akt inhibitor. Interestingly, CD8+ T cells in these Akt-cultures were inhibited in their differentiation stage, expressing higher levels of CD45RA and CCR7 compared to controls. In addition, expression of CD95, IL2Rβ, and IL7Rα was also elevated confirming the TSCM-like phenotype. Although proliferation of the Akt-inhibited CD8+ T cells was decreased as shown by less PBSE dilution, expansion could be significantly preserved. Next, we investigated whether the established culture conditions could be used to generate MiHA-specific TSCM-like cells. Therefore, CD8+T cells from MiHA-negative donors were primed using autologous MiHA peptide-loaded moDCs in the presence of the Akt-inhibitor. Interestingly, MiHA-specific T cell priming could be induced, consisting of mainly TCMand TSCM-like cells compared to almost entirely TEMcells in the control setting. Akt-inhibited MiHA-specific T cells showed higher expression of CCR7, CD45RA, CD62L, CD28, CD95, and IL7Rα. Importantly, for the Akt-inhibited MiHA-specific T cells, proliferation was reserved, resulting in robust proliferation capacity during restimulation after removal of the Akt-inhibitor. The resulting TEFFcells were highly functional, showing capacity to degranulate and produce IFNγ upon peptide restimulation. In conclusion, by inhibiting the Akt-pathway, in vitroCD8+ T cell differentiation can be reduced. Therefore, Akt signalling inhibition can be exploited for generating TSCM-like MiHA-specific T cells in adoptive immunotherapy after allo-SCT.

Tordoir: Glycostem Therapeutics: Employment. Spanholtz:Glycostem Therapeutics: Employment.