Immunotherapy Of Leukemia Research Articles

Advances of Research on Application of Leukemia Xenotransplantation Model--Review

Abstract Tumor xenograft model (PDTX) derived from leukemia patients is an animal model in which the leukemia cells or primary cell lines of patients are transplanted directly into immunodeficient mice.The emergence of nude mice and SCID mice opened early xenotransplantation, then the NSG, NOG mice and the improved model and humanized mice based on there mice significantly improves the success rate of transplantation. The late presented transplantation of leukemia LSC and transplantation of patient-derived and induced pluripotent stem cells obtained based on iPSC technology provide new insight for the anderstanding leukemia genesis and development, and the new type humanized mouse model with normal lymphatic hematopoietic reconstruction provides a new platform of leukemia cell therapy and immunotherapy for leukemia therapy. PDTX is an important platform for the study of the pathogenesis and drug resistance mechanism of leukemia, as well as the development of new drugs and individualized treatment. In this paper, the recent progress in the construction and application of models of immunodeficient mice and their models is reviewed.

Abstract 679: Quantitative systems pharmacology modeling of immunotherapies in B-cell acute lymphoblastic leukemia

Abstract Introduction: Multiple T-cell engaging antibody-based molecules and CAR-T therapies are in development for the treatment of B-cell acute lymphoblastic leukemia (B-ALL). Moreover, two therapies have already been approved by FDA: blinatumomab (CD19/CD3 bispecific T-cell engager) and tisagenlecleucel (anti-CD19 CAR-T therapy). This work employs quantitative systems pharmacology (QSP) modeling approach to investigate factors affecting outcomes of administration of such types of therapy. Methods: Model was developed on the basis of Immune Response Template (QSP model of immune system). It describes leukemic blasts, hematopoiesis of normal B cells, CD4+ and CD8+ T-cell subsets, and cytokines (IL-6, TNFα, IFNγ, TGFβ, etc.) in bone marrow and blood. The model takes into account the different level of expression of CD19 on various subsets of normal B cells and leukemic cells. Model parameters were estimated and fitted against in vitro, in vivo human data. Small part of clinical data on blinatumomab and anti-CD19 CAR-T therapies was used for model calibration, other part was used for model validation. Results: Our model reproduced clinically relevant blinatumomab and anti-CD19 CAR-T hallmark data including depletion of CD19+ cells, T-cell subset dynamics and cytokine release post infusion. The model successfully described the clinically observed phenomenon on progressive cytokine release attenuation with subsequent blinatumomab treatment cycles. It confirmed the dose step strategy for decreasing initial cytokine release. Model showed that treatment outcome is very sensitive to target expression, level of immune cells, disease progression rate and expression of PD-L1 on leukemic cells. The proportion of CD19+ and CD19- leukemic blasts before treatment can be used as a biomarker to predict probability of relapse in patients to stratify patients. Conclusions: This work provides a quantitative modeling framework to predict responses to immunotherapies in B-ALL on the basis of realistic baseline conditions and reveal the factors affecting treatment outcome. Citation Format: Oleg Demin Jr, Antonina Nikitich, Oleg Demin. Quantitative systems pharmacology modeling of immunotherapies in B-cell acute lymphoblastic leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 679.

Abstract 3371: Mapping the evolution of T cell states during DLI response and resistance using single-cell data and computational tools

Abstract Donor lymphocyte infusion (DLI) is a standard of care and potentially curative immunotherapy for relapsed leukemia after allogeneic hematopoietic stem cell transplant (allo-SCT). Despite low response rates for many leukemias, chronic myelogenous leukemia (CML) has historically exhibited an exquisite DLI sensitivity, and we previously reported that durable response to DLI was associated with reversal of exhaustion of bone marrow (BM) -infiltrating T cells (Bachireddy et al., Blood 2014). Critical questions remain, however, regarding the exact transcriptional states of those T cell subtypes mediating exhaustion, anti-leukemia responses, and resistance to DLI. To map evolving phenotypic T cell states in situ at single cell resolution, we profiled viable cells isolated from cryopreserved BM mononuclear cells from a median of 3 timepoints before and after DLI from 12 patients with relapsed CML after allo-SCT, including 6 long-term responders to DLI (R’s) and 6 nonresponders (NR’s), using single cell RNA sequencing (scRNA-seq). Using our computational tools for processing and analyzing scRNA-seq data (Azizi et al., Cell 2018), we detected 381,462 cells in total derived from 43 unique patient-timepoints that met our quality metrics. Because DLI’s anti-leukemic efficacy derives in large part from T cell activity, we sought a more refined characterization of T cells using our tool Biscuit (Azizi et al., Cell 2018) to merge, normalize and cluster T cells. We observed a marked increase in the number of T cell clusters in post-DLI samples compared to matched pre-DLI samples (p<0.001). Both R and NR cases exhibited increases in phenotypic volume induced by DLI (p<1x10-6), suggesting DLI induces multiple, independent gene expression components in both clinical outcomes. However, at both pre- and post-DLI timepoints, phenotypic volumes in R cases were higher than that of NR cases. Comparing T cell exhaustion between R vs NR cases, we confirmed increased T cell exhaustion signatures in R-pre T cells. Using factor analysis techniques we found that anergy, dysfunction and tolerance are shared factors driving a subset of T cells that are enriched in NRs, suggesting multiple forms of T cell dysfunction in DLI resistance. We found that the clusters dominated by post-DLI R T cells were characterized by greater diversity of T helper subsets (Th1, Tfh, Th2, Th9, and Th22) and enrichment for exhaustion, type I and II IFN pathways, proinflammatory gene sets and CD8 T cell activation. Clusters dominated by NR T cells displayed increases in Th17 and Treg signatures, anergy and tolerance. These data suggest that (1) pretreatment T cell phenotypic diversity may be important for DLI response; (2) that DLI increases such diversity differently in R’s than in NR’s; (3) while T cell subsets exhibit some overlap pre-therapy, responders and non-responders become increasingly dissimilar post therapy. Citation Format: Elham Azizi, Pavan Bachireddy, Vinhkhang N. Nguyen, Shuqiang Li, Donna S. Neuberg, Robert J. Soiffer, Jerome Ritz, Edwin P. Alyea III, Dana Pe'er, Catherine J. Wu. Mapping the evolution of T cell states during DLI response and resistance using single-cell data and computational tools [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3371.

CSPG4-Specific CAR T Cells for High-Risk Childhood B Cell Precursor Leukemia.

The advent of CD19-specific chimeric antigen receptor (CAR) T cells has proven to be a powerful asset in the arsenal of cancer immunotherapy of acute lymphoblastic leukemia and certain B cell lymphomas. However, a sizable portion of patients treated with CD19-CAR T cells relapse with CD19-negative cancer cells, necessitating the quest for back-up antigens. Chondroitin sulfate proteoglycan 4 (CSPG4) expression has been reported on leukemic blasts bearing the ill-fated MLL 11q23 rearrangement. We aimed at exploring the use of CSPG4-specific CAR T cells against mixed-lineage leukemia (MLL)-rearranged leukemic blasts, using the precursor B cell leukemia cell line KOPN8 (MLL–MLLT1 translocation) as a model. First, we confirmed CSPG4 expression on KOPN8 cells. Bulk T cells electroporated with mRNA encoding a CSPG4-specific CAR upregulated activation markers and secreted the Th1 cytokines TNF and IFNγ in an antigen-specific manner upon co-culture with KOPN8 cells. More importantly, CSPG4-specific CAR T cells evinced specific degranulation towards KOPN8 cells and specifically lysed KOPN8 target cells in chromium lysis experiments. CSPG4 is a well-established CAR target in cutaneous melanoma. Here, we provide proof-of-principle data for the use of CSPG4-specific CAR T cells against MLL-translocated leukemias.

S910 A NEW MEMBRANE‐BOUND IL‐21/4–1BBL‐EXPANDED NK PRODUCT EXHIBITS ACTIVITY AGAINST ACUTE MYELOID LEUKEMIA ACTIVITY IN VIVO

Background:Adoptive NK cells infusion is a promising immunotherapy for acute myeloid leukemia (AML) patients.Aims:The aim of this study was to explore that two weeks of clinical grade expansion could provide sufficient numbers and purity of NK cells for clinical application with an enhanced ant‐leukemia response in vitro.Methods:NK cells were expanded in the large‐scale expansion platform based on membrane‐bound interleukin‐21/4–1BBL K562 feeder cells (mbIL21/4‐1BBL). The phenotype as well as responsitveness of NKcells were evaluated before and post NK cells expansion. NCG mouse model were used to assess the in vivo persistence and in vivo anti‐leukemia effect of NK products. Finally, 20 minimal residual disease persistent or relapse (MRD+) AML were enrolled to evaluate the safety of adoptive expanded NK products infusion.Results:In this study, using the large‐scale expansion platform, we first demonstrated that 2 weeks of expansion could satisfy clinical use in number (∼4.1 × 109 NK cell) and purity (median 81%) from 2 × 107 peripheral blood mononuclear cells based on membrane‐bound interleukin‐21/4‐1BBL K562 feeder cells (mbIL21/4‐1BBL), with expansion folds of 1318 and 2876 at 2 and 3 weeks, respectively. The CD107α production as well as TNF‐α secretion capacity of NK products against AML cell lines, or primary AML blasts, were comparable between 2 weeks and 3 weeks expanded NK products, both of which were higher than those resting NK cells before expansion. The MbIL21/4‐1BBL expanded NK product xenografted into immunodeficient leukemia mice could be found in bone marrow, spleen, liver, lung and peripheral blood, persisting for at least 2 weeks and reducing the AML burden in vivo. NK cells infusions once, or three times every other days exhibited similar tumor control. Compared with the case‐paired 20 minimal residual disease persistent or relapse (MRD+) AML patients with regular consolidation therapy, 20 AML MRD+ patients who accepted the expanded NK treatment showed better leukemia‐free survival and overall survival.Summary/Conclusion:In conclusion, this study demonstrated that mbIL‐21/4‐1BBL expanded NK cells exhibited anti‐leukemia activity against AML in vitro and in a mouse model, and the infusion of expanded NK product was potentially efficacious for AML patient without adverse effects.

PF207 TARGETING WILMS TUMOR 1 WITH A T CELL BISPECIFIC ANTIBODY (WT1‐TCB): EX VIVO AND IN VIVO POTENCY BY BIVALENT RECOGNITION OF PEPTIDE‐MHC COMPLEXES FROM AN INTRACELLULAR TUMOR ANTIGEN

Background:Antibody‐based immunotherapy represents a promising strategy to target chemo‐resistant leukemic cells. However, classical antibody‐based approaches are restricted to cell surface antigens, even though targeting of intracellular antigens would allow further access to a large number of tumor‐specific target structures.Aims:In this study we evaluated a 2+1 T Cell Bispecific (TCB) antibody based on T cell receptor (TCR)‐like antibodies for immunotherapy of acute myeloid leukemia (AML). The TCB targets the intracellular tumor antigen Wilms Tumor 1 (WT1) by bivalent recognition of the peptide RMFPNAPYL in the context of human leukocyte antigen allele A∗02:01 (HLA‐A2). Complementary binding to CD3ε recruits T cells irrespective of their TCR‐specificity.Methods:WT1 expression levels in cancer cell lines and primary AML patient samples at different time points during course of the disease were determined by quantitative real‐time PCR, western blot and immunohistochemical staining. WT1‐TCB‐mediated cytotoxicity was analyzed by co‐cultivation of WT1‐expressing HLA‐A2+ cancer cell lines with T cells from healthy donors. Specific lysis was assessed by flow cytometry. TCR downstream signaling was measured by co‐cultivation of primary AML cells with NFAT Luciferase Reporter Jurkat cells. WT1‐TCB‐mediated cytotoxicity against primary AML cells was evaluated in our pre‐established feeder layer‐based ex vivo long‐term culture system. For in vivo testing, NSG mice (NOD.Cg‐Prkdcscid‐Il2rgtm1Wjl/SzJ) were humanized with human HLA‐A2+ CD34+ cord blood cells. After successful engraftment and development of human T cells, WT1‐expressing HLA‐A2+ SKM‐1 tumor cells were subcutaneously inoculated followed by weekly administration of the WT1‐TCB.Results:In accordance with previous reports, we observed WT1 expression in 79% (n = 38) of cancer cell lines and in 92% (n = 65) of AML patient samples at the time of initial diagnosis. Moreover, WT1 expression levels correlated with the percentage of AML blasts: no significant WT1 expression was observed at time of CR (n = 26), whereas WT1 was expressed again at time of relapse (n = 21). WT1‐TCBs elicited antibody‐mediated T cell cytotoxicity against peptide‐pulsed T2 cells and AML cell lines in a WT1 and HLA‐restricted manner. Equally, TCR downstream signaling was observed in a WT1‐restrictive manner by co‐cultivation of primary AML cells with NFAT Luciferase Reporter Jurkat cells. WT1‐TCBs further mediated specific lysis of primary AML cells upon addition of allogenic T cells from healthy donors (mean specific lysis: 67 ± 6% after 13–14 days; ± SEM; n = 18). Correspondingly, up‐regulation of T cell activation and surrogate exhaustion markers was observed (MFI fold change CD69: 9.3 ± 1.5, PD‐1: 5.1 ± 0.7, TIM‐3: 4.7 ± 0.6; ± SEM; n = 22). WT1‐TCBs also mediated killing of primary AML cells in an autologous setting (mean specific lysis: 38 ± 13% after 13–14 days; ± SEM; n = 5). In comparison with WT1RMF‐specific T cells, only bivalent binding by WT1‐TCB induced efficient lysis of primary AML cells. Furthermore, WT1‐TCB‐treated humanized mice bearing SKM‐1 tumors showed a dose dependent and significant reduction in tumor growth resulting in tumor control.Summary/Conclusion:TCR‐like TCBs targeting of intracellular tumor antigens are a promising tool for cancer immunotherapy. Notably, the 2+1 TCB molecular format for bivalent binding facilitates potent in vitro, ex vivo and in vivo killing of AML cell lines and primary AML samples which present low numbers of the RMF peptide‐MHC complex on the cell surface validating WT1‐TCB as a promising therapeutic agent for the treatment of AML.

PF217 TARGETING FLT3 IN AML: MODULATION OF FLT3‐BITE® ACTIVITY THROUGH COMBINATION WITH VARIOUS TKI

Background:T cell recruiting antibody constructs represent a novel platform for immunotherapy of acute myeloid leukemia (AML). We previously characterized FLT3 (CD135; FMS‐like tyrosine kinase 3) as a suitable target antigen for treatment of AML based on significantly higher expression on primary AML (pAML) cells in comparison to healthy hematopoietic subpopulations (Lindl ASH 2017).Aims:Here, we evaluated T cell‐redirected target cell lysis mediated by a FLT3 BiTE® antibody construct (FLT3 BiTE®) against AML cells in an ex vivo model for pAML cells, an in vivo NOD‐SCID mouse admixture model and in non human primates (NHP). We hypothesized that combining FLT3 BiTE® with a tyrosine kinase inhibitor (TKI) would enhance target cell lysis. Accordingly, FLT3 expression, mutational status, and TKI‐mediated T‐cell dysfunction were analyzed as relevant factors for FLT3 BiTE®‐mediated cytotoxicity.Methods:FLT3 BiTE®‐mediated cytotoxicity was evaluated by coculture of various FLT3+ AML cell lines with healthy donor (HD) T cells. Cytotoxicity against pAML cells and T‐cell proliferation were tested ex vivo using a long‐term culture system (Krupka Blood 2014) and assessed by flow cytometry. The Effector:Target cell ratio was based on residual T cells within the sample.In vivo, a NOD‐SCID mouse admixture model with human CD3+ T cells and MOLM‐13 cells was used. In NHP, FLT3 BiTE® was administered with step‐dosing at 5, 15, 45, and 100 μg/kg/d in a 16‐day continuous intravenous study. Pharmacodynamics were determined by measuring blood (PB) and bone marrow (BM) FLT3 transcript and soluble FLT3 ligand.TKI‐mediated modulation of T‐cell function was assessed by CD3/CD28‐bead stimulation of CFSE‐labeled HD T cells. Cytotoxicity of 3 TKIs against AML cell lines with different FLT3‐ITD mutational status was evaluated.Last, the combination of TKI with FLT3 BiTE® was tested in cytotoxicity assays using HD T cells with FLT3‐ITD+ AML cell lines or pAML cells.Results:FLT3 BiTE® exhibited cytotoxicity against FLT3+ AML cell lines at picomolar concentrations irrespective of FLT3 mutational status (EC50:1.9 ± 0.7pM, ± SD, n = 6). No cytotoxicity was observed against FLT3− cell lines. pAML cells were lysed (%specific lysis day 9: 43 ± 9%, ± SEM, n = 14) even at low E:T ratios (1:3–1:74) accompanied by strong T‐cell proliferation (fold change CD2+ day 9: 5.9 ± 1.8, ± SEM) independent of FLT3 mutational status. In mice treated intraperitoneally with 200 μg/kg/d FLT3 BiTE®, reduction in tumor volume was observed (tumor volume d15 FLT3 BiTE® vs control BiTE®: 221 vs 1431 mm3, p≤0.0001, n = 10 per group). Furthermore, a decrease in FLT3 transcript levels (d17: 92.1 ± 0.1%, ± SD, n = 3) as well as a time‐dependent increase in circulating soluble FLT3 ligand levels were observed in PB and BM samples from NHP, supporting a FLT3 BiTE®‐mediated decrease in FLT3+cells.Pharmacologically relevant concentrations of TKIs induced cytotoxicity against FLT3‐ITD+ AML cell lines (Table, top) without altering T‐cell proliferation. Higher concentrations of TKI led to a decrease in T‐cell proliferation (Table, middle).Next, we combined TKIs with FLT3 BiTE®, resulting in increased cytotoxicity against FLT3‐ITD+ AML cell lines (p = 0.03, n = 6) as well as pAML cells in 2 FLT3‐ITD+ patients (Table, bottom).Summary/Conclusion:The FLT3 BiTE® antibody construct showed strong cytotoxicity against FLT3+ cells in vitro and ex vivo, mediated antitumor activity and depleted FLT3+ cells in vivo. The addition of clinically relevant concentrations of TKI enhanced FLT3‐BiTE®‐mediated cytotoxicity in FLT3‐ITD+ AML while higher TKI concentrations compromised T‐cell activity.image

A skewed distribution and increased PD-1+Vβ+CD4+/CD8+ Tcells in patients with acute myeloid leukemia.

The limited application of immunotherapy in acute myeloid leukemia (AML) may be due to poor understanding of the global Tcell immune dysfunction in AML. In this study, we analyzed the distribution characteristics of 24 TCR Vβ subfamilies in CD3+, CD4+, and CD8+ Tcells in AML patients and healthy controls. The percentage of TCR Vβ subfamily Tcells was predominately lower in most AML cases, while it was increased in some cases. TCR Vβ2+Tcells were increased in AML, particularly TCR Vβ2+CD4+Tcells, which were significantly higher. To further address the immunosuppression in different Vβ subfamilies, we characterized the distribution of program death-1 (PD-1)+Tcells in TCR Vβ subfamilies of CD4+ and CD8+Tcells. Significantly higher levels of PD-1+Vβ+Tcells were found for most Vβ subfamilies in most AML cases. A higher percentage of PD-1+Vβ2+Tcells with a high number of Vβ2+Tcells was found in all of the CD3+, CD4+, and CD8+ Tcell subsets. Moreover, increasing PD-1+Vβ7.2, Vβ8+, Vβ14+, Vβ16+, and Vβ22+CD8+Tcells were distributed in the AML-M5 subtype group compared with the AML-M3 group. In addition, higher PD-1+ Vβ5.2+ and PD-1+ Vβ12+CD8+Tcells were associated with AML patients who had a poor response to chemotherapy. In conclusion, increased PD-1+Vβ+Tcells is a common characteristic of AML, higher PD-1+Vβ2+Tcells may be associated with a low antileukemia effect, and higher PD-1+Vβ5.2+ and PD-1+Vβ12+CD8+Tcells may be related to poor prognosis in AML. These characteristics may be worth considering as immune biomarkers for clinical outcome in AML.

Hemap: An Interactive Online Resource for Characterizing Molecular Phenotypes across Hematologic Malignancies.

Large collections of genome-wide data can facilitate the characterization of disease states and subtypes, permitting pan-cancer analysis of molecular phenotypes and evaluation of disease context for new therapeutic approaches. We analyzed 9,544 transcriptomes from more than 30 hematologic malignancies, normal blood cell types, and cell lines, and showed that disease types could be stratified in a data-driven manner. We then identified cluster-specific pathway activity, new biomarkers, and in silico drug target prioritization through interrogation of drug target databases. Using known vulnerabilities and available drug screens, we highlighted the importance of integrating molecular phenotype with drug target expression for in silico prediction of drug responsiveness. Our analysis implicated BCL2 expression level as an important indicator of venetoclax responsiveness and provided a rationale for its targeting in specific leukemia subtypes and multiple myeloma, linked several polycomb group proteins that could be targeted by small molecules (SFMBT1, CBX7, and EZH1) with chronic lymphocytic leukemia, and supported CDK6 as a disease-specific target in acute myeloid leukemia. Through integration with proteomics data, we characterized target protein expression for pre-B leukemia immunotherapy candidates, including DPEP1. These molecular data can be explored using our publicly available interactive resource, Hemap, for expediting therapeutic innovations in hematologic malignancies. SIGNIFICANCE: This study describes a data resource for researching derailed cellular pathways and candidate drug targets across hematologic malignancies.

Natural Products and Acute Myeloid Leukemia: A Review Highlighting Mechanisms of Action.

Recent findings have shown great potential of alternative interventions such as immunotherapy and natural products for acute myeloid leukemia (AML). This study aims to review the anti-AML effect of various natural compounds. Natural compounds were classified into five groups: alkaloids, carotenoids, nitrogen-containing compounds, organosulfur compounds or phenolics based on each compound’s chemical properties. Fifty-eight studies were collected and reviewed in this article. Phenolics are the most abundant group to have an apoptotic effect over AML cells, while other groups have also shown significant apoptotic effects. Some compounds induced apoptosis by regulating unique mechanism like human telomerase reverse transcriptase (hTERT) or laminin receptor (67LR), while others modified caspases, poly (adp-ribose) polymerase (PARP) and p53. Further study is required to identify side-effects of potent compounds and the synergistic effects of combination of two or more natural compounds or existing conventional anti-AML drugs to treat this dreadful disease.

Interleukin-15 in cancer immunotherapy: IL-15 receptor complex versus soluble IL-15 in a cancer cell-delivered murine leukemia model

Abstract Cancer immunotherapy endeavors to stimulate the immune system in order to recognize, reject and destroy tumor cells. Previously we have demonstrated that IL-12 is an ideal candidate for leukemia immunotherapy and there is a Phase 1 human trial underway at the Princess Margaret Cancer Center to test this approach in AML patients. In order to further our knowledge on immunomodulators we have expanded our studies to other cytokines such as IL-15. IL-15, either in its soluble form (IL-15sol) or complexed with IL-15Ra (IL-15Rc), has been shown to exhibit potent anti-tumor activities in various cancer studies. In this study we describe the impact of intraperitoneal IL-15 in a cancer cell delivered IL-15 immunotherapy approach using the 70Z/3-L leukemia mouse model. Whereas both forms of IL-15 led to significantly improved survival rates compared to the parent cell line, there were striking differences in the extent of the improved survival: mice receiving cancer cells secreting IL-15sol showed near 100% survival for up to 250 days, whereas mice receiving cancer cells secreting IL-15Rc started to perish around day 50 post-injection, with few survivors left on day 250. Interestingly, cell-secreted IL-15sol and IL-15Rc activated different cell types in the peritoneum, leading to different immune responses. IL-15Rc resulted in an influx and/or expansion of NK1.1+ natural killer cells in the peritoneum, while injection of cells secreting IL-15sol lead to an increased expansion of CD4+ and CD8+ T-cells. To our knowledge, this is the first study detailing a significantly different biological effect of cell-delivered IL-15Rc vs IL-15sol in a mouse cancer immunotherapy study, suggesting a different mechanism of action of the two IL-15 forms.

Human CAR NK Cells: A New Non-viral Method Allowing High Efficient Transfection and Strong Tumor Cell Killing.

CAR-NK cells may represent a valuable tool, complementary to CAR-T cells, in adoptive immunotherapy of leukemia and solid tumors. However, gene transfer to human NK cells is a challenging task, particularly with non-virus-based techniques. Here, we describe a new procedure allowing efficient electroporation-based transfection of plasmid DNA, including CAR and CCR7 genes, in resting or cytokine-expanded human NK cell populations and NK-92 cell line. This procedure may offer a suitable platform for a safe and effective use of CAR-NK cells in adoptive immunotherapy of cancer.

Dendritic Cell-Based Immunotherapy of Acute Myeloid Leukemia.

Acute myeloid leukemia (AML) is a type of blood cancer characterized by the uncontrolled clonal proliferation of myeloid hematopoietic progenitor cells in the bone marrow. The outcome of AML is poor, with five-year overall survival rates of less than 10% for the predominant group of patients older than 65 years. One of the main reasons for this poor outcome is that the majority of AML patients will relapse, even after they have attained complete remission by chemotherapy. Chemotherapy, supplemented with allogeneic hematopoietic stem cell transplantation in patients at high risk of relapse, is still the cornerstone of current AML treatment. Both therapies are, however, associated with significant morbidity and mortality. These observations illustrate the need for more effective and less toxic treatment options, especially in elderly AML and have fostered the development of novel immune-based strategies to treat AML. One of these strategies involves the use of a special type of immune cells, the dendritic cells (DCs). As central orchestrators of the immune system, DCs are key to the induction of anti-leukemia immunity. In this review, we provide an update of the clinical experience that has been obtained so far with this form of immunotherapy in patients with AML.

Recent Advances in Immunotherapy for Acute Myeloid Leukemia --Review

Currently, the traditional chemotherapeutic drugs for acute myeloid leukemia (AML) showed significant curative efficacy, including the improving long-term prognosis and the life quality of the patients, however the traditional chemotherapeuatic drugs showed the some limitatious in the aspects of enhancing the complete remission rate of newly diagnosed AML patients, overcoming the relapse after remission,as well as the primary and secondary drug-resstance to chemotherapeutics. In order to improve the long-term prognisis of patients, the immuno therapy will the best choice for these patients. This review sammarizes the main current advance of immunotherapy of AML at home and abroad, such as antibody-drug conjugate(ADC), bispecific T cell engager (BiTE), chimeric type of antigen receptor T cell (CAR-T) therapy, checkpoint inhibitors; dendritic cell vaccination and peptide vaccines; natural killer cell (NK) therapy and so on.

The HLA-B −21 dimorphism impacts on NK cell education and clinical outcome of immunotherapy in acute myeloid leukemia

AbstractNatural killer (NK) cell function is regulated by inhibitory receptors, such as the family of killer immunoglobulin-like receptors (KIRs) and the NKG2A/CD94 heterodimer. These receptors recognize cognate HLA class I molecules on potential target cells, and recent studies imply that an HLA-B dimorphism at position −21 in the gene segment encoding the leader peptide dictates whether NK cell regulation primarily relies on the KIRs or the NKG2A/CD94 receptor. The impact of this HLA-B dimorphism on NK cell–mediated destruction of leukemic cells or on the course of leukemia is largely unknown. In a first part of this study, we compared functions of NK cells in subjects carrying HLA-B −21M or 21T using interleukin-2 (IL-2)–activated NK cells and leukemic cells from patients with acute myeloid leukemia (AML). Subjects carrying HLA-B −21M harbored better-educated NKG2A+ NK cells and displayed superior capacity to degranulate lytic granules against KIR ligand-matched primary leukemic blasts. Second, we aimed to define the potential impact of HLA-B −21 variation on the course of AML in a phase 4 trial in which patients received IL-2–based immunotherapy. In keeping with the hypothesis that 21M may be associated with improved NK cell functionality, we observed superior leukemia-free survival and overall survival in −21M patients than in −21T patients during IL-2–based immunotherapy. We propose that genetic variation at HLA-B −21 may determine the antileukemic efficacy of activated NK cells and the clinical benefit of NK cell–activating immunotherapy.

Antigen Targets for the Development of Immunotherapies in Leukemia.

Immunotherapeutic approaches, including allogeneic stem cell transplantation and donor lymphocyte infusion, have significantly improved the prognosis of leukemia patients. Further efforts are now focusing on the development of immunotherapies that are able to target leukemic cells more specifically, comprising monoclonal antibodies, chimeric antigen receptor (CAR) T cells, and dendritic cell- or peptide-based vaccination strategies. One main prerequisite for such antigen-specific approaches is the selection of suitable target structures on leukemic cells. In general, the targets for anti-cancer immunotherapies can be divided into two groups: (1) T-cell epitopes relying on the presentation of peptides via human leukocyte antigen (HLA) molecules and (2) surface structures, which are HLA-independently expressed on cancer cells. This review discusses the most promising tumor antigens as well as the underlying discovery and selection strategies for the development of anti-leukemia immunotherapies.

Role of natural killer cells for immunotherapy in chronic myeloid leukemia (Review).

The majority of natural killer (NK) cells serve an important role in eliminating malignant cells. The cytotoxic effects of NK cells were first identified against leukemia cells, and it is now hypothesized that they may have a critical role in leukemia therapy. The cellular functions of NK cells are mediated by their cell surface receptors, which recognize ligands on cancer cells. The role of NK cells is specifically regulated by the activating or inhibitory killer cell immunoglobulin‑like receptors (KIRs) on their surface, which bind to the human leukocyte antigen (HLA) class I ligands present on the target cells. The association between KIR and HLA is derived from the diversity of KIR/HLA gene profiles present in different individuals, and this determines the cytotoxic effect of NK cells on cancer cells. Chronic myeloid leukemia (CML) is a hematological leukemia characterized by the hyper‑proliferation of myeloid cells, with the majority of patients with CML presenting with abnormal immune cells. Tyrosine kinase inhibitors are the present standard therapy for CML, but are associated with numerous adverse side effects. Various studies have proposed CML therapy by immunotherapeutic approaches targeting the immune cells. This review summarizes the contents of NK cells and the association between KIR/HLA and leukemia, especially CML. This is followed by a discussion on the development of NK cell immunotherapy in hematological malignancies and research into strategies to enhance NK cell function for CML treatment.

Increased detection of (leukemiaspecific) adaptive and innate immune-reactive cells under treatment of AML-diseased rats and one therapy-refractory AML-patient with blastmodulating, clinically approved response modifiers

Background: Kits (combinations of immune-modulatory compounds: GM-CSF+Picibanil (Kit-I), Prostaglandine (PG-E2,Kit-K) or +PGE1 (Kit-M), patent 102014014993) convert myeloid blasts into dendritic cells of leukemic origin (DCleu). After stimulation with DCleu, antileukemic T-cells can be generated ex vivo. Compounds are approved for clinical use and are attractive tools for immunotherapy in myeloid leukemia.

Abstract A044: Mutated NPM1 as target for immunotherapy of acute myeloid leukemia

Abstract The most frequent subtype of acute myeloid leukemia (AML) is defined by mutations in the nucleophosmin (NPM1) gene. Mutated NPM1 is an attractive target for immunotherapy, since it is an essential driver gene and 4 base pair frameshift insertions in exon 12 occur in 30-35% of AML, resulting in a novel C-terminal alternative reading frame of 11 amino acids. By searching in the HLA class I ligandome of primary AML, we identified multiple peptides derived from mutated NPM1. For one of these peptides, i.e., HLA-A*02:01-presented CLAVEEVSL, we searched for specific T-cells in AML patients and healthy individuals using peptide-MHC tetramers. Tetramer-positive CD8 T-cell clones were isolated and analyzed for reactivity against primary AML with mutated NPM1. From one selected clone with superior antitumor reactivity, we isolated the T-cell receptor (TCR) and demonstrated specific recognition and lysis of HLA-A*02:01-positive AML with mutated NPM1 in vitro after retroviral transfer to CD8 and CD4 T-cells. In vivo antitumor efficacy of TCR-transduced CD8 and CD4 T-cells was confirmed in immunodeficient mice engrafted with a human AML cell line expressing mutated NPM1. These data show that mutated NPM1-derived peptides are presented on AML and that CLAVEEVSL is a neoantigen that can be efficiently targeted on AML with mutated NPM1 by TCR gene transfer in a co-receptor independent fashion. Immunotherapy targeting mutated NPM1 may therefore contribute to treatment of AML. Citation Format: Dyantha I. van der Lee, Rogier M. Reijmers, M. Willy Honders, Renate S. Hagedoorn, Rob. M. de Jong, Michel G.D. Kester, Dirk M. van der Steen, Arnoud H. de Ru, Christiaan Kweekel, Inge Jedema, Hendrik Veelken, Mirjam M. Heemskerk, Peter A. van Veelen, J.H. Frederik Falkenburg, Marieke Griffioen. Mutated NPM1 as target for immunotherapy of acute myeloid leukemia [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A044.

Progress of immunotherapy of acute myelogenous leukemia

Acute myelogenous leukemia(AML) is a highly heterogeneous malignant hematologic disease, and its clinical treatment mainly includes traditional chemotherapy, but the efficacy is limited and the patients with worse performance status and comorbidities can not be treated with chemotherapy. In recent years, immunotherapy has achieved certain curative effects in AML which shows a promising prospect. This review introduces immunotherapy of AML reported in 60th American Society of Hematology(ASH) Annual Meeting. Key words: Leukemia, myelogenous, acute; Immunotherapy