Application of NK Cells in ASCT Peritransplantation of Multiple Myeloma

Abstract BACKGROUND Significant progress has been made in the therapy of multiple myeloma (MM) with the autologous hematopoietic stem cell transplantation(ASCT) in recent years. But the relapse after transplantation is still the main problem because the grafts are often contaminated by tumor cells and have no effect of anti myeloma. The residual myeloma cells are reduced by increasing doses of cytotoxic drug in the pretreatment. But the related mortality of ASCT is increased because of side effects and complications. Therefore, the pretreatment of MM hasn't changed very much in recent 50 years. So it is urgent to find a new pretreatment to improve curative effect which has small side effects. Natural killer (NK) cells are the principal effector cells in the innate immune system. They have extensive biological functions including anti-tumor and antiviral effects, etc. There is no report about applying NK cells as a pretreatment for ASCT to treat MM so far. In this study, NK cells were added to the pretreatment of ASCT to treat MM We hope NK cells can increase the curative effect and reduce relapse rate, but not increase the side effects. Meanwhile, they can clear myeloma cells without killing by chemotherapy drugs and to decrease infection in transplantation. OBJECTIVE To observe the safety and effectiveness of the technology by applying NK cells in peritransplantation of MM ASCT. METHODS The ages of 8 MM patients including 7 males and 1 female were 42-62 years, the median age was 51 years, 2 patents of CR and 6 patents of PR before transplantation. The method of NK cell culture: membrane embedded active factor culture system. The pretreatment mainly based on standard Melphalan or busulfan. NK cells were infused to 2 cases back 6-7days after infusing stem cell, and to 6 cases back 24-48 hours before infusing stem cells and 48 hours after chemotherapy as a part of pretreatment in transplantation. Stem cell infusion volume: the number of CD34+ cell 2.2-4.0×106/kg, the number of mononuclear cells 4.3-5.8×106/kg. NK cell infusion volume: 16-160×106/kg. we observed the adverse reactions, recovery time of blood cells and the curative effects. RESULTS There were no instances of fever, rash, diarrhea,shock, and other adverse reactions in this study. Recovery time of hematopoietic cells was: 7-14d of granulocyte and 9d of the median time, 9-34d of platelet and 11d of the median time. Fever(38-39 degrees) occurred in 6 patients during the inhibitory period of bone marrow after ASCT. Infection is the major cause of fever in patients receiving ASCT. We evaluated therapeutic effect 3 months after transplantation was 3 cases of CR and 5 cases of PR in 8 patients. The effect 6 months after transplantation was 3 cases of CR and 3 cases of PR in 6 patients. 8 patients were given thalidomide for maintenance therapy. The follow-up time was 3-29 months, median follow-up time was 11 months. Up to the present, there was no death case. CONCLUSIONS It was safe and effective to infuse autologous NK cells back in peritransplantation of MM ASCT. Storm effect of inflammatory factors didn't occur during this study. We will expand the number of cases for further clinical research to observe the long-term therapeutic effects and adverse reactions. Disclosures No relevant conflicts of interest to declare.

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Pluripotent Stem Cell-Derived Human Natural Killer Cells with Potent Anti-Multiple Myeloma Activity,

Blood ◽

10.1182/blood.v118.21.4034.4034 ◽

2011 ◽

Vol 118 (21) ◽

pp. 4034-4034

Author(s):

David A. Knorr ◽

Zhenya Ni ◽

Allison Bock ◽

Vijay G. Ramakrishnan ◽

Shaji Kumar ◽

...

Keyword(s):

Stem Cells ◽

Multiple Myeloma ◽

Stem Cell ◽

Nk Cells ◽

Natural Killer ◽

Tumor Cells ◽

Adoptive Immunotherapy ◽

Target Cell ◽

Peripheral Blood ◽

Nk Cell

Abstract Abstract 4034 Natural Killer (NK) cells are lymphocytes of the innate immune system with anti-viral and anti-cancer activity. Over the past decade, they have gained interest as a promising cellular source for use in adoptive immunotherapy for the treatment of cancer. Most notably, NK cells play an important role in the graft-vs-tumor effect seen in allogeneic hematopoietic stem cell transplantation (allo-HSCT), and a better understanding of NK cell biology has translated into improved transplant outcomes in acute myelogenous leukemia (AML). Small studies have demonstrated a role for NK cell activity in multiple myeloma (MM) patients receiving allo-HSCT. Investigators have also utilized haplo-identical killer immunoglobulin-like receptor (KIR) mismatched NK cells for adoptive immunotherapy in patients with multiple myeloma (MM). Our group has focused on the development of NK cells from human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) as a novel starting source of lymphocytes for immunotherapy. We have previously demonstrated potent anti-tumor activity of hESC-derived NK cells in vitro and in vivo against a variety of different targets. We have also shown that iPSC-derived NK cells from a variety of different somatic cell starting sources posses potent anti-tumor and anti-viral activity. Here, we demonstrate hESC- and iPSC-derived NK cell development in a completely defined, feeder-free system that is amenable to clinical scale-up. These cultures contain a pure population of mature NK cells devoid of any T or B cell contamination, which are common adverse bystanders of cellular products isolated and enriched from peripheral blood. Our cultures are homogenous for their expression of CD56 and express high levels of effector molecules known to be important in anti-MM activity, including KIR, CD16, NKG2D, NKp46, NKp44, FasL and TRAIL. We have now tested the activity of hESC- and iPSC-derived NK cells against MM tumor cells in order to provide a universal source of lymphocytes for adoptive immunotherapy in patients with treatment refractory disease. We find that similar to peripheral blood NK cells (PB-NK), hESC- and iPSC-derived NK cells are cytotoxic against 3 distinct MM cell lines in a standard chromium release cytotoxicity assay. Specifically, activated PB-NK cells killed 48.5% of targets at 10 to 1 effector to target ratios, whereas hESC (46.3%) and iPSC (42.4%) derived NK cells also demonstrated significant anti-MM activity. Also, hESC- and iPSC-derived NK cells secrete cytokines (IFNγ and TNFα) and degranulate as demonstrated by CD107a surface expression in response to MM target cell stimulation. When tested against freshly isolated samples from MM patients, hESC- and IPSC-derived NK cells respond at a similar level as activated PB-NK cells, the current source of NK cells used in adoptive immunotherapy trials. These MM targets (both cell lines and primary tumor cells) are known to express defined ligands (MICA/B, DR4/5, ULBP-1, BAT3) for receptors expressed on NK cells as well as a number of undefined ligands for natural cytotoxicity receptors (NCRs) and KIR. As these receptor-ligand interactions drive the anti-MM activity of NK cells, we are currently evaluating expression of each of these molecules on the surface of both the effector and target cell populations. Not only do hESC- and iPSC-derived NK cells provide a unique, homogenous cell population to study these interactions, they also provide a genetically tractable source of lymphocytes for improvement of the graft-vs-myeloma effect and could be tailored on a patient specific basis using banks of hESC-or iPSC-derived NK cells with defined KIR genotypes for use as allogeneic or autologous effector cells. Disclosures: No relevant conflicts of interest to declare.

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NK Cell Reconstitution After Autologous Hematopoietic Stem Cell Transplantation: Association Between NK Cell Maturation Stage and Outcome in Multiple Myeloma

Frontiers in Immunology ◽

10.3389/fimmu.2021.748207 ◽

2021 ◽

Vol 12 ◽

Author(s):

Ane Orrantia ◽

Iñigo Terrén ◽

Gabirel Astarloa-Pando ◽

Carmen González ◽

Alasne Uranga ◽

...

Keyword(s):

Multiple Myeloma ◽

Stem Cell ◽

Hematopoietic Stem Cell Transplantation ◽

Stem Cell Transplantation ◽

Nk Cells ◽

Cell Transplantation ◽

Hematopoietic Stem Cell ◽

Nk Cell ◽

Cell Maturation ◽

Hematopoietic Stem

Autologous hematopoietic stem cell transplantation (autoHSCT) is a standard of care for transplant-eligible patients with multiple myeloma (MM). Among factors that influence outcome after autoHSCT, it has been suggested that the number of natural killer (NK) cells plays an important role. However, the impact that different NK cell subsets and their phenotype could have in disease progression after autoHSCT are less clear. For this reason, we have phenotypically and functionally characterized NK cells during immune system reconstitution after autoHSCT in 54 MM patients. Shortly after leukocyte recovery, an extensive redistribution of NK cell subsets occurs in these patients. In addition, NK cells undergo a profound phenotypic change characterized, among others, by their increased proliferative capacity and immature phenotype. Importantly, MM patients who showed lower frequencies of the mature highly differentiated NKG2A-CD57+ NK cell subset at +30 and +100 days after autoHSCT experienced superior progression-free survival and had a longer time to the next treatment than those with higher frequencies. Our results provide significant insights into NK cell reconstitution after autoHSCT and suggest that the degree of NK cell maturation after autoHSCT affects the clinical outcome of MM patients treated with this therapeutic strategy.

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Proteasome Inhibitor Bortezomib Disrupts Tumor Necrosis Factor-Related Apoptosis-Inducing ligand (TRAIL) Expression and Natural Killer (NK) Cell Killing of TRAIL Receptor-Positive Multiple Myeloma Cells

Blood ◽

10.1182/blood.v116.21.5021.5021 ◽

2010 ◽

Vol 116 (21) ◽

pp. 5021-5021

Author(s):

Xiaoli Feng ◽

Jie Yan ◽

Yibiao Wang ◽

Juleen R. Zierath ◽

Magnus Nordenskjöld ◽

...

Keyword(s):

Multiple Myeloma ◽

Nk Cells ◽

Proteasome Inhibitor ◽

Tumor Necrosis ◽

Nk Cell ◽

Dependent Manner ◽

Trail Expression ◽

Myeloma Cells ◽

Dose Dependent ◽

Necrosis Factor

Abstract Abstract 5021 Bortezomib, a potent 26S proteasome inhibitor, is approved for the treatment of multiple myeloma (MM) and clinical trials are under way to evaluate its efficacy in other malignant diseases. However, cytotoxic effects of bortezomib on immune-competent cells have also been observed. The aim of this study was to investigate the putative effects of Bortezomib on activated human natural killer (NK) cell function. In this project, primary lymphocytes or NK cells were isolated or purified from peripheral blood mononuclear cells of healthy donors. Expression of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) by interleukin (IL)-2-activated NK cells was quantified by flow cytometry and real time-PCR method. NK cell degranulation activity and cytotoxicity against MM cell lines were evaluated by CD107a surface translocation and 51Cr release assays, respectively. Our results demonstrate that bortezomib markedly downregulated cell surface expression of TRAIL on primary human IL-2-activated NK cells in a dose-dependent manner. Moreover, bortezomib reduced TRAIL mRNA expression in these cells, suggesting that bortezomib regulates TRAIL expression of activated NK cells at the transcriptional level. Interestingly, pharmacological inhibition of the transcription factor NF-κB also profoundly decreased TRAIL expression both at protein and mRNA levels, indicating a novel role of NF-κB in the regulation of TRAIL expression in activated human NK cells. Furthermore, perforin-independent killing of the human MM cell lines was significantly suppressed following bortezomib treatment. In addition, blocking cell surface-bound TRAIL with a TRAIL antibody impaired lymphokine-activated killer (LAK) cell-mediated lysis of the TRAIL-sensitive MM cell line, RPMI8226. Our study demonstrated that bortezomib decreases TRAIL expression by IL-2 activated NK cells in a dose-dependent manner and disrupts TRAIL-mediated killing of TRAIL-sensitive myeloma cells, suggesting that Bortezomib may potentially hamper NK-dependent immunosurveillance against tumors in patients treated with this drug. Disclosures: No relevant conflicts of interest to declare.

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CD80-IL2 Expressing Myeloma Cells for Immune Gene Therapy of Multiple Myeloma

Blood ◽

10.1182/blood.v118.21.4718.4718 ◽

2011 ◽

Vol 118 (21) ◽

pp. 4718-4718

Author(s):

Giulia Giunti ◽

David Malone ◽

Lucas Chan ◽

Darling David ◽

Shahram Y Kordasti ◽

...

Keyword(s):

Multiple Myeloma ◽

T Cells ◽

T Cell ◽

Nk Cells ◽

Nk Cell ◽

Cytolytic Activity ◽

Immune Gene ◽

Moderate Increase ◽

Myeloma Cells ◽

Cell Functions

Abstract Abstract 4718 Improved experimental therapies are needed for Multiple Myeloma (MM). Despite major progress in treatment and initial induction of remission, myeloma remains an incurable disease. Although immunotherapy and, in particular, the employment of NK cells offers an approach of interest for the treatment of Multiple Myeloma (MM), recent studies have shown that myeloma cells utilise a number different mechanisms to impair NK and T cell functions. Important amongst these mechanisms is the reduced expression of CD80 in the sub-populations of PBMC isolated from myeloma patients. We have previously demonstrated CD80/IL-2 mediated stimulation of NK and T cells isolated from AML patients (as measured by proliferation, cytokine release and target cell specific cytolytic activity). In the present study we have examined the ability of genetically modified MM cells engineered to express CD80 and IL-2 to stimulate NK cell functions. These studies confirm the ability of MM cells to suppress NK cell functions in healthy PBMC and show that in contrast to the unmodified MM cells, the CD80/IL-2 expressing MM cells are able to stimulate a moderate increase in NK and T cell numbers and a significant increase in the fraction of NK cells with activatory receptors (NKp44, NKp30, NKp46) and activation markers (CD69) on the cell surface of both NK and T cells. More importantly for potential therapeutic applications the stimulated NK cells show increased cytolytic activity against the unmodified MM cells. This data suggest that CD80/IL-2 MM cells may be able to overcome the immune suppressive functions of unmodified MM cells and to stimulate NK, and T cell mediated responses against the unmodified MM cells. Therefore CD80/IL-2 expressing MM cells may provide a suitable cellular vaccine for NK cell stimulation and possibly the induction of broader ranging immunological responses against multiple myeloma cells. Disclosures: No relevant conflicts of interest to declare.

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ATM-ATR–dependent up-regulation of DNAM-1 and NKG2D ligands on multiple myeloma cells by therapeutic agents results in enhanced NK-cell susceptibility and is associated with a senescent phenotype

Blood ◽

10.1182/blood-2008-08-173914 ◽

2009 ◽

Vol 113 (15) ◽

pp. 3503-3511 ◽

Cited By ~ 247

Author(s):

Alessandra Soriani ◽

Alessandra Zingoni ◽

Cristina Cerboni ◽

Maria Luisa Iannitto ◽

Maria Rosaria Ricciardi ◽

...

Keyword(s):

Multiple Myeloma ◽

Nk Cells ◽

Nk Cell ◽

Ex Vivo ◽

Therapeutic Agents ◽

Therapeutic Drug ◽

Similar Data ◽

Nkg2d Ligand ◽

Myeloma Cells ◽

Nkg2d Ligands

Abstract There is much evidence to support a role for natural killer (NK) cells in controlling the progression of multiple myeloma (MM), a malignancy characterized by an abnormal plasma cell proliferation in the bone marrow (BM). Induction of DNA damage response has been recently shown capable of enhancing NKG2D ligand (NKG2DL) expression, but nothing is known about DNAM-1 ligand (DNAM-1L) regulation. In this study, we show that myeloma cells treated with low doses of therapeutic agents commonly used in the management of patients with MM, such as doxorubicin, melphalan, and bortezomib, up-regulate DNAM-1 and NKG2D ligands. Accordingly, therapeutic drug treatment of MM cells increases NK-cell degranulation, the NKG2D and DNAM-1 receptors being the major triggering molecules. Similar data were also obtained using ex vivo primary plasma cells derived from MM patients. Drug-induced DNAM-1 and NKG2D ligand expression was abolished after treatment with the ATM (ataxia telangiectasia mutated) and ATR (ATM- and RAD3-related) pharmacologic inhibitors caffeine and KU-55933, and was preferentially associated with senescent cells arrested in the G2 phase of the cell cycle. Altogether, our findings have identified a common pathway that can trigger the up-regulation of different NK cell–activating ligands and suggest that NK cells represent an immunosurveillance mechanism toward cells undergoing stress-induced senescent programs.

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Natural killer cells efficiently target multiple myeloma clonogenic tumor cells

Cancer Immunology Immunotherapy ◽

10.1007/s00262-021-02901-y ◽

2021 ◽

Author(s):

Alejandra Leivas ◽

Ruth M. Risueño ◽

Alma Guzmán ◽

Laura Sánchez-Vega ◽

Manuel Pérez ◽

...

Keyword(s):

Multiple Myeloma ◽

Nk Cells ◽

Nk Cell ◽

Side Population ◽

Cell Activity ◽

Nk Cell Activity ◽

Self Renewal ◽

Myeloma Cells ◽

Nkg2d Receptor ◽

Clonogenic Cells

AbstractThe multiple myeloma (MM) landscape has changed in the last few years, but most patients eventually relapse because current treatment modalities do not target clonogenic stem cells, which are drug-resistant and can self-renew. We hypothesized that side population (SP) cells represent myeloma clonogenic stem cells and, searching for new treatment strategies, analyzed the anti-myeloma activity of natural killer (NK) cells against clonogenic cells. Activated and expanded NK cells (NKAE) products were obtained by co-culturing NK cells from MM patients with K562-mb15-41BBL cell line and characterized by flow cytometry. Functional experiments against MM cells were performed by Eu-TDA release assays and methylcellulose clonogenic assays. Side population was detected by Dye Cycle Violet labeling and then characterized by flow cytometry and RNA-Seq. Self-renewal capacity was tested by clonogenic assays. Sorting of both kind of cells was performed for time-lapse microscopy experiments. SP cells exhibited self-renewal potential and overexpressed genes involved in stem cell metabolism. NK cells from MM patients exhibited dysregulation and had lower anti-tumor potential against clonogenic cells than healthy donors’ NK cells. Patients’ NK cells were activated and expanded. These cells recovered cytotoxic activity and could specifically destroy clonogenic myeloma cells. They also had a highly cytotoxic phenotype expressing NKG2D receptor. Blocking NKG2D receptor decreased NK cell activity against clonogenic myeloma cells, and activated NK cells were able to destroy SP cells, which expressed NKG2D ligands. SP cells could represent the stem cell compartment in MM. This is the first report describing NK cell activity against myeloma clonogenic cells.

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CD38-Deficient, CD16-Engineered NK Cells Exhibit Enhanced Antibody-Dependent Cellular Cytotoxicity without NK Cell Fratricide to Augment Anti-Myeloma Immunity in Combination with Daratumumab

Blood ◽

10.1182/blood-2018-99-116459 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 3224-3224

Author(s):

Karrune Woan ◽

Ryan Bjordahl ◽

Frank Cichocki ◽

Svetlana Gaidarova ◽

Cameron Pride ◽

...

Keyword(s):

Multiple Myeloma ◽

Cell Death ◽

Nk Cells ◽

Peripheral Blood ◽

Research Funding ◽

Nk Cell ◽

Cellular Cytotoxicity ◽

Antibody Dependent Cellular Cytotoxicity ◽

Myeloma Cells ◽

Cell Immunotherapy

Abstract Daratumumab targets the cell surface protein CD38 and is the only FDA approved monoclonal antibody that has demonstrated single agent efficacy in relapsed refractory myeloma. CD38 is broadly expressed in the immune system, and its high expression on multiple myeloma cells allows for effective targeting by daratumumab. Daratumumab induces myeloma cell death through multiple mechanisms, including complement-dependent cytotoxicity, antibody-dependent cellular phagocytosis, and perhaps most importantly, antibody-dependent cellular cytotoxicity (ADCC). ADCC is mediated by binding of the antibody Fc region to the CD16 Fc receptor expressed on natural killer (NK) cells. Engagement of CD16 induces NK cell activation and target cell cytolysis. However, because CD38 is also expressed on the surface of NK cells, daratumumab treatment can induce NK cell fratricide, which likely impairs the effectiveness of ADCC-mediated targeting and elimination of myeloma. In addition, NK cell function is often suppressed or absent in patients with myeloma, as a result of the tumor itself or from its therapy, further reducing the effectiveness of daratumumab. Collectively, preclinical and clinical observations suggest a potential therapeutic benefit of maintaining NK cell numbers and function in patients to support daratumumab-mediated ADCC and augment the treatment of multiple myeloma. We have developed an off-the-shelf NK cell immunotherapy derived from genetically engineered, induced pluripotent stem cells (iPSC) for enhanced ADCC in combination with daratumumab. iPSCs were engineered to express a high-affinity, non-cleavable CD16 construct (hnCD16) in combination with complete bi-allelic disruption of the CD38 gene (hnCD16 CD38-/-), and the engineered iPSCs were subsequently differentiated into NK (iNK) cells. We hypothesized that CD38-deficient iNK cells would exhibit improved survival by avoiding daratumumab-induced NK cell fratricide, while expression of the hnCD16 transgene would enhance ADCC against myeloma cells in combination with daratumumab. Genetic modification was confirmed in hnCD16 CD38-/- iNK cells by flow cytometry, demonstrating abrogation of CD38 expression (Fig. 1A) and constitutive high expression of CD16 (Fig. 1B). Additionally, hnCD16 iNK cells and hnCD16 CD38-/- iNK cells expressed similar levels of SLAMF7/CD319 (the target of elotuzumab) and NKG2A (Fig. 1C and D). No significant difference in iNK cell differentiation, expansion, maturation, activation, or ability to mediate natural cytotoxicity was observed. In contrast to previous reports, we observed no effect of CD38-deficiency on CD16-mediated calcium flux between hnCD16 iNK cells and hnCD16 CD38-/- iNK cells (Figure 1E). In vitro culture of NK cells in the presence of daratumumab led to NK cell fratricide for both peripheral blood-derived NK cells and hnCD16 iNK cells (Fig. 1F). Daratumumab-induced NK cell fratricide was dependent upon expression of both CD16 and CD38, as unmodified iNK with low CD16 levels (~20% of cells) showed reduced cell death in the presence of daratumumab, which was entirely absent in hnCD16 CD38-/- iNK cells (Fig. 1F). This data was confirmed by extended culture of NK cells with RPMI-8226 tumor spheroids in the presence or absence of daratumumab. The number of hnCD16 iNK cells and peripheral blood NK cells were significantly reduced compared to hnCD16 CD38-/- iNK cells (p>0.005, Fig. 1 G). Importantly, hnCD16 CD38-/- iNK cells were better able to mediate ADCC towards MM1.S multiple myeloma cells compared to hnCD16 iNK cells (Fig. 1H). Taken together, these data support our hypothesis that targeted knock out of CD38 on NK cells alleviates daratumumab-induced NK cell fratricide that occurs through the crosslinking of CD16 and CD38 on neighboring NK cells, leading to augmented anti-myeloma immunity. These data provide a translatable, proof of concept study demonstrating precision genetic engineering of iPSC to generate off-the-shelf NK cell immunotherapy to enhance daratumumab mediated ADCC in multiple myeloma. We propose a strategy of off-the-shelf hnCD16 CD38-/- iNK infusion in combination with daratumumab to overcome NK cell depletion effects of CD38 targeted agents and to improve myeloma patient outcomes. Figure 1. Figure 1. Disclosures Bjordahl: Fate Therapeutics Inc.: Employment. Cichocki:Fate Therapeutics Inc.: Consultancy, Research Funding. Gaidarova:Fate Therapeutics Inc: Employment. Pride:Fate Therapeutics Inc.: Employment. Kaufman:Fate Therapeutics: Consultancy, Research Funding. Malmberg:Fate Therapeutics Inc.: Consultancy, Research Funding. Valamehr:Fate Therapeutics Inc.: Employment.

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Phase I Trial of IL-21 Ex Vivo Expanded NK Cells Administration to Prevent Disease Relapse after Haploidentical Stem-Cell Transplantation for Myeloid Leukemias

Blood ◽

10.1182/blood.v126.23.102.102 ◽

2015 ◽

Vol 126 (23) ◽

pp. 102-102 ◽

Cited By ~ 2

Author(s):

Stefan O. Ciurea ◽

Dean A. Lee ◽

Kai Cao ◽

Gabriela Rondon ◽

Julianne Chen ◽

...

Keyword(s):

Stem Cell ◽

Stem Cell Transplantation ◽

Phase I ◽

Nk Cells ◽

Nk Cell ◽

Ex Vivo ◽

Disease Relapse ◽

Post Transplant ◽

Equity Ownership

Abstract Background: As outcomes of haploidentical stem cell transplantation (HaploSCT) have improved, disease relapse represents the most common cause of treatment failure. Methods: We initiated a phase I clinical trial (clinicaltrials.gov NCT01904136) using peripheral blood-derived NK cells expanded ex vivo for 14 days with K562 antigen presenting cells expressing membrane-bound (mb) IL21 to prevent disease relapse after HaploSCT for patients with myeloid malignancies (AML,CML,MDS). We hypothesized that infusion of expanded and activated NK cells would compensate for the lower NK cell function in the early post-transplant period, and higher NK cell numbers would enhance anti-tumor effects of the graft. The primary endpoints were safety and determining the maximum tolerated dose (MTD). Patients were treated with a melphalan-based conditioning regimen (Figure). All patients had a primary bone marrow graft. NK cells were generated from peripheral blood mononuclear cells of the same donor obtained prior to marrow harvest and infused on days -2, +7 and on/after +28. The first infusion was with fresh and the other two were with cryopreserved NK cells. The dose escalation was planned in cohorts of 2 patients starting at 1x105/kg up to 1x109/kg or more if MTD will not be reached. Predictive NK alloreactivity and/or donor KIR B genotyping was preferred but not required to participate on study, however, was evaluated in all patients (Table). Results: Ten patients have been enrolled and treated to date. Of these, 8 patients were beyond Day+30 and were evaluated, 5 with AML (3 in CR1 with intermediate and 1 with high-risk cytogenetics, and 1 in CR2 FLT3+ with persistent MRD by flow cytometry) and 3 with CML (2 in second chronic phase, one with clonal evolution who failed multiple TKIs). The median age was 39 years (range 18-59). Four patients were males and 4 females. The NK cells dose escalation was as follows: 1x105/kg (N=2), 1x106/kg (N=3) and 1x107/kg (N=2). One patient was treated with 1x104/kg before full evaluation of 1x105/kg was completed. All patients achieved primary engraftment (100%). All patients except the one who received the lowest dose (1x104/kg) had sustained engraftment and 100% donor chimerism on Day 30 post-transplant. The median time to neutrophil engraftment was 18 days and to platelet engraftment was 26 days. The first patient had a mixed chimerism, developed secondary graft failure and concurrent parainfluenza pneumonia. He was re-transplanted with a different donor but died of treatment-related mortality (TRM). Of 7 patients evaluable for aGVHD, the maximum aGVHD grade was gr II in 4 patients. No gr III-IV aGVHD or cGVHD was observed. Only 3/7 patients had CMV reactivation (43% compared with 71% in retrospective data with the same treatment without NK cells), 2 requiring a brief period of treatment of approximately 1 month. None developed BK virus hemorrhagic cystitis. All patients achieved CR after transplant. One patient (#2) treated at 1x105/kg NK cell dose relapsed, received salvage treatment and is alive at last follow-up. All other patients are alive and in remission (N=6) after a median follow-up of 6 months (range 1-12.5). NK cell phenotype and function early post-transplant will be presented at the meeting. Conclusions: Doses up to 1x107/kg of ex vivo expanded NK cells using the mbIL-21 method can be safely administered after HaploSCT. Administration of these cells in this setting in not associated with a higher incidence of aGVHD. There was a low rate of viral reactivation, suggesting that the infused NK cells may provide antiviral activity. MTD has not been reached, the study is ongoing. Table. PT Nr Initials NK cell dose (/kg) Pt KIR Ligand Donor Donor KIR Ligand NK Allo-reactivity Donor KIR Haplotype # Cen-B/B KIR Score KIR Centromeric KIR2DS1 Outcome 1 RB 1x104 C2/C2, Bw4 Son C2, Bw4 No A/A 0 Neutral Cen-A/A - Died 2 FM 1x105 C1/C2, Bw4 Son C1, Bw4 No A/B 2 Better Cen-A/B - Relapsed +120 3 RG 1x105 C1/C2, Bw4 Daughter C1, C2, Bw4 No A/A 0 Neutral Cen-A/A - CR +374 4 GM 1x106 C1/C1, Bw4 Sister C1, C2, Bw4 Yes A/B 2 Better Cen-A/B - CR +168 5 DS 1x106 C1/C1 Brother C1, C2, Bw4 Yes A/A 0 Neutral Cen-A/A - CR +166 6 MH 1x107 C1/C2, Bw4 Sister C1, Bw4 No A/B 2 Best Cen-B/B + CR +91 7 JG 1x106 C1/C2, Bw4 Sister C1, C2, Bw4 No A/A 0 Neutral Cen-A/A - CR +35 8 DD 1x107 C1/C1, Bw4 Father C1, Bw4 No A/A 0 Neutral Cen-A/A - CR +87 9 RR 3x107 C1/C1 Brother C1, C2 Yes A/B 2 Better Cen-A, Cen/Tel-B - NE 10 JC 3x107 C2/C2, Bw4 Son C1/C2, Bw4 Yes A/B 2 Better Cen-A/B + NE Figure 1. Figure 1. Disclosures Lee: Intrexon: Equity Ownership; Ziopharm: Equity Ownership; Cyto-sen: Equity Ownership. Rezvani:Pharmacyclics: Research Funding.

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Early CMV Replication-Associated Anti-Leukemia Effect Is Related to the Reconstitution of Specific Subsets of Natural Killer Cells after Haploidentical Stem Cell Transplantation in Acute Leukemia

Blood ◽

10.1182/blood-2018-99-115733 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 2128-2128

Author(s):

Jieun Jang ◽

Haerim Chung ◽

Yu Ri Kim ◽

Hoi-kyung Jeung ◽

Ju-In Eom ◽

...

Keyword(s):

Stem Cell ◽

Viral Load ◽

Acute Leukemia ◽

Nk Cells ◽

Cell Transplantation ◽

Relapse Rate ◽

Nk Cell ◽

Cmv Infection ◽

Cmv Reactivation

Abstract Background: Cytomegalovirus (CMV) infection is a major infectious complication after allogeneic hematopoietic cell transplantation. Recently, it has been reproducibly demonstrated that CMV reactivation is associated with decreased relapse rate in patients with acute myeloid leukemia (AML). The aim of this study is to evaluate the impact of early CMV reactivation on the incidence of disease relapse in relation to the reconstitution of subsets of natural killer (NK) cells after haploidentical stem cell transplantation (haploSCT) in acute leukemia. Methods: Clinical data from 47 adult patients diagnosed with acute leukemia who underwent their first haploSCT between September 2009 and December 2017 was retrospectively analyzed. All patients were unable to find a suitable HLA-matched donor in their families or donor registries. Patient blood samples were collected prior to conditioning therapy and following haploSCT at day 30 and day 90. Peripheral blood mononuclear cells obtained from 28 patients abundant cells at every time points were analyzed for flow cytometric immunophenotyping. Expression of specific receptors (NKG2A, NKG2C, NKG2D, DNAM1 and NKp46) on the NK cells (CD56brightCD16- or CD56dim/-CD16+ cells) were serially quantified by multiparametric flow cytometry using appropriate monoclonal antibodies. Results: Median age was 38 years (range, 21-62 years), and 28 (54%) patients were male. Thirty-six (69%) patients received a transplant in their first complete remission (CR) status. Median follow-up duration was 54 months (range, 6.6-83.3 months), and all patients were CMV seropositive before receiving a transplant. Early CMV replication occurred at a median of 23 days after haploSCT in 40 of 47 patients (85%). Among these patients, 14 had more than two episodes of CMV replication throughout the follow-up period. The median peak viral load during CMV replication was 54,000 copies/mL. In univariate analysis, early CMV replication (P < 0.001), older donor age (P = 0.018), high dose of infused T cells (P = 0.022) and chronic graft-versus-host disease (GVHD, P = 0.001) were significantly associated with lower 3-year cumulative incidence of relapse after haploSCT. Early CMV replication was correlated with higher leukemia-free survival (LFS, P < 0.001). In multivariate analysis, early CMV replication (hazard ratio [HR], 0.24; 95% confidence interval [CI], 0.060 to 0.930, P = 0.039) and chronic GHVD (HR, 0.25; CI, 0.089 to 0.695; P = 0.008) were identified as independent factors for higher LFS rate. CMV reactivation was associated with higher overall survival (OS) rates and increased nonrelapse mortality, although there was no statistical significance. The viral load at the initiation of CMV-specific treatment was significantly associated with OS rates. Patients with CMV viral load higher than 45,000 copies/mL had lower OS rate compared to those with lower CMV load (34.5% versus 89.5%, P = 0.022). Longitudinal analysis of NK cell reconstitution after haploSCT showed that the CMV infection was associated with the increased expansion of CD56brightCD16dim/- NK cell, particularly in DNAM1+ NK cell subset. The rate of CD56brightCD16dim/-DNAM1+ NK cells increment was significantly higher in the patients with CMV infection compared with patients without CMV infection (P = 0.022). Importantly, we observed that the cumulative relapse rate was significantly decreased in patients with an increased CD56brightCD16dim/- DNAM1+ NK cells compared to patients with low CD56brightCD16dim/- DNAM1+ NK cells (20.4% versus 63.6% , P = 0.016). Conclusion: Early CMV replication was identified as an independent prognostic factor for LFS in acute leukemia in the haploSCT setting. Increased reconstitution of CD56brightCD16dim/- DNAM1+ NK cells was associated with CMV infection-related reduction in the relapse rate. Further studies are required to elucidate the anti-leukemia effects of these NK cell subsets associated with CMV infection in haploSCT. Disclosures Kim: Novartis Korea: Honoraria.

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Treatment of Myeloid and Lymphoid Malignancies with Highly Purified Alloreactive CD56+CD3− NK-Cells and Haploidentical Stem Cell Transplantation: Promising Results of a Phase I Study.

Blood ◽

10.1182/blood.v106.11.2906.2906 ◽

2005 ◽

Vol 106 (11) ◽

pp. 2906-2906 ◽

Cited By ~ 1

Author(s):

Chiara Gentilini ◽

Urte Hilbers ◽

Goetz Hartung ◽

Thoralf Lange ◽

Constanze Kliem ◽

...

Keyword(s):

Stem Cell ◽

Stem Cell Transplantation ◽

Nk Cells ◽

Cell Transplantation ◽

Nk Cell ◽

Good Condition ◽

Leukemic Cells ◽

Hematopoietic Stem ◽

Technical Problems

Abstract NK cell alloreactivity can mediate strong graft versus leukemia (GvL) effects following haploidentical hematopoietic stem cell transplantation (HSCT). In an attempt to further improve the antileukemic effectiveness of this approach, we have adoptively transferred high numbers of alloreactive donor NK cells during the early phase after transplantation. Method: In a phase-II study, 10 patients (6 AML, 1 MDS, 1 HD, 1 CML, 1 ALL, median age 38 yrs, range 17–48 yrs) were transplanted in late phases of their disease (5 pts. as 2nd transplantation) and received purified NK cells from their haploidentical donors at day +2 after HSCT. Conditioning consisted of 12 Gy fTBI, Thiotepa (10mg/kg), Fludarabine (5 x 30 mg/qm) and OKT3 (day −4 to +2). Two patients received a reduced conditioning with Fludarabine and OKT3 alone. NK cells were isolated from the CD34- fraction using an automated two-step procedure of CD3+ depletion and subsequent CD56+ selection. Results: No severe technical problems occurred and a mean of 12,1 x 10E8 (16,74 x10E6/kg, range 6,12 to 27,2 x10E6/kg) CD34+ cells was selected in high purity (95,9 %) with a very low T-cell content (mean 1,83 x10E4/kg CD3+ cells, 4,5 Log depletion). A mean of 5,7 x10E8 (6,7 x 10E6/kg) CD56+CD3− NK cells was transferred (yield 70,36%, purity 70%). The mean number of contaminating CD3+ cells was 3,2 x10E4/kg (3,6 Log depletion).No severe acute toxicity attributable to NK cell infusion was observed. Hematopoietic recovery was fast with leukocytes > 1/nl between day 5 and 11. Seven patients developed early grade II GvHD of the skin which promptly resolved after CSA and steroids. One patient developed late graft rejection five months after reduced conditioning, received a 2nd graft from a different donor, engrafted but unfortunately died due to pneumonia one month after the second transplantation. One pt with CML died due to adenovirus infection at day 140. One pt. with HD in 5th CR died due to pneumonia at day 85. One patient showed a relapse of the AML three months after transplant. She received a DLI with NK cells form the donor but died due to disease progression one month after. Interestingly, leukemic cells from this patient proved to be resistant to donor NK cell mediated lysis. Five of 9 patients are alive and in CCR with a median follow up of 192 days, the two patients with the longest follow up are in very good condition and free of GvHD at day +1271 and +1019. Our data show for the first time that the early adoptive transfer of high numbers of HLA-mismatched NK cells is safe and feasible.

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