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Successes with anti-CD20 antibodies in chronic lymphocytic leukemia (CLL) and enhanced activity of Fc-engineered vs unmodified antibody therapy suggest a potentially impactful role for natural killer (NK) cells and other innate immune cells in controlling this disease. Stimulated NK cells have shown promise as a cellular therapy, but their application has been constrained by limited expansion capacity and low cytotoxic activity against CLL cells. Here, we demonstrate that both healthy donor-derived and CLL patient-derived NK cells expand rapidly when stimulated with feeder cells expressing membrane-bound interleukin-21 (mbIL-21) and have potent cytotoxic activity against allogeneic or autologous CLL cells. Combination with anti-CD20 antibodies significantly enhances NK recognition and killing of CLL targets. As any CLL immune therapy would likely be given in combination, we assess commonly used treatments and demonstrate that ibrutinib has mixed suppressive and protective effects on expanded NK cells, whereas expanded NKs are highly resistant to venetoclax. We demonstrate efficacy in vivo in 2 xenograft mouse models of human CLL that support building upon a regimen of venetoclax and obinutuzumab with mbIL-21–expanded NK cells. Collectively, these data support development of mbIL-21–expanded NKs combined with the CD20 antibody obinutuzumab and venetoclax in the treatment of CLL.

Promising results have been reported for adult patients with high-risk hematologic malignancies undergoing haploidentical bone marrow transplant (haploBMT) with post-transplantation cyclophosphamide (PTCy). We report results from the first multicenter trial for pediatric and young adult patients with high-risk acute leukemias and myelodysplastic syndrome (MDS) in the US and Canada in the Pediatric Transplantation and Cellular Therapy Consortium (PTCTC). Nine centers transplanted 32 patients with acute leukemias or MDS with myeloablative conditioning (MAC), haploBMT with PTCy, mycophenolate mofetil, and tacrolimus. The median age was 12y (range 1-23y). Diagnoses included acute myeloid leukemia (n=15), acute lymphoid leukemia (n=11), mixed lineage leukemia (n=1), and MDS (n=5). Transplant-related mortality (TRM) at 180 days, our primary objective, was 0%. The cumulative incidence (CuI) of acute graft-versus-host disease (aGVHD) grade II at Day 100 was 13%. No patients developed aGVHD grades III-IV. The CuI of moderate-severe chronic GVHD (cGVHD) at 1 year was 4%. Donor engraftment occurred in 27/32 (84%). Primary graft failures included three patients that received suboptimal bone marrow grafts, all successfully engrafted after second transplants. The CuI of relapse at 1y was 32%, with more relapse in pre-BMT MRD+ versus MRD- patients. Overall survival at 1y and 2y is 77% and 73%, and event-free survival at 1y and 2y is 68% and 64%, respectively. We demonstrate no TRM or severe aGVHD, low cGVHD, and favorable relapse and survival rates. This successful pilot has led to a phase III trial comparing MAC haploBMT versus HLA-matched unrelated donors, now open in the Children's Oncology Group.

Supplementary Figures and Legends

Clinical response rates after adoptive cell therapy (ACT) are highly correlated with in vivo persistence of the infused T cells. However, antigen-specific T cells found in tumor sites are often well-differentiated effector cells with limited persistence. Central memory CD8+ T cells, capable of self-renewal, represent desirable ACT products. We report here that exposure to a histone deacetylase inhibitor (HDACi) and IL21 could reprogram differentiated human CD8+ T cells into central memory–like T cells. Dedifferentiation of CD8+ T cells was initiated by increased H3 acetylation and chromatin accessibility at the CD28 promoter region. This led to IL21-mediated pSTAT3 binding to the CD28 region, and subsequent upregulation of surface CD28 and CD62L (markers of central memory T cells). The reprogrammed cells exhibited enhanced proliferation in response to both IL2 and IL15, and a stable memory-associated transcriptional signature (increased Lef1 and Tcf7). Our findings support the application of IL21 and HDACi for the in vitro generation of highly persistent T-cell populations that can augment the efficacy of adoptively transferred T cells.

Natural killer (NK) cells are useful for cancer immunotherapy and have proven clinically effective against hematologic malignancies. However, immunotherapies for poor prognosis solid malignancies, including ovarian cancer, have not been as successful due to immunosuppression by solid tumors. Although rearming patients' own NK cells to treat cancer is an attractive option, success of that strategy is limited by the impaired function of NK cells from cancer patients and by inhibition by self-MHC. In this study, we show that expansion converts healthy donor and immunosuppressed ovarian cancer patient NK cells to a cytotoxic CD56superbrightCD16+ subset with activation state and antitumor functions that increase with CD56 brightness. We investigated whether these expanded NK cells may overcome the limitations of autologous NK cell therapy against solid tumors. Peripheral blood- and ascites-derived NK cells from ovarian cancer patients were expanded and then adoptively transferred into cell-line and autologous patient-derived xenograft models of human ovarian cancer. Expanded ovarian cancer patient NK cells reduced the burden of established tumors and prolonged survival. These results suggest that CD56bright NK cells harbor superior antitumor function compared with CD56dim cells. Thus, NK cell expansion may overcome limitations on autologous NK cell therapy by converting the patient's NK cells to a cytotoxic subset that exerts a therapeutic effect against autologous tumor. These findings suggest that the value of expanded autologous NK cell therapy for ovarian cancer and other solid malignancies should be clinically assessed. Cancer Immunol Res; 6(10); 1174–85. ©2018 AACR.

Expression of NK cell activation and inhibitory ligands on resistant primary ovarian cancer cells.

Purpose: To activate and propagate populations of γδ T cells expressing polyclonal repertoire of γ and δ T-cell receptor (TCR) chains for adoptive immunotherapy of cancer, which has yet to be achieved.Experimental Design: Clinical-grade artificial antigen-presenting cells (aAPC) derived from K562 tumor cells were used as irradiated feeders to activate and expand human γδ T cells to clinical scale. These cells were tested for proliferation, TCR expression, memory phenotype, cytokine secretion, and tumor killing.Results: γδ T-cell proliferation was dependent upon CD137L expression on aAPC and addition of exogenous IL2 and IL21. Propagated γδ T cells were polyclonal as they expressed TRDV1, TRDV2-2, TRDV3, TRDV5, TRDV7, and TRDV8 with TRGV2, TRGV3F, TRGV7, TRGV8, TRGV9*A1, TRGV10*A1, and TRGV11 TCR chains. IFNγ production by Vδ1, Vδ2, and Vδ1negVδ2neg subsets was inhibited by pan-TCRγδ antibody when added to cocultures of polyclonal γδ T cells and tumor cell lines. Polyclonal γδ T cells killed acute and chronic leukemia, colon, pancreatic, and ovarian cancer cell lines, but not healthy autologous or allogeneic normal B cells. Blocking antibodies demonstrated that polyclonal γδ T cells mediated tumor cell lysis through combination of DNAM1, NKG2D, and TCRγδ. The adoptive transfer of activated and propagated γδ T cells expressing polyclonal versus defined Vδ TCR chains imparted a hierarchy (polyclonal>Vδ1>Vδ1negVδ2neg>Vδ2) of survival of mice with ovarian cancer xenografts.Conclusions: Polyclonal γδ T cells can be activated and propagated with clinical-grade aAPCs and demonstrate broad antitumor activities, which will facilitate the implementation of γδ T-cell cancer immunotherapies in humans. Clin Cancer Res; 20(22); 5708–19. ©2014 AACR.

PDF file - 1405KB, Supplementary Materials and Methods. Supplementary Figures - (1) Tumor cell line culture conditions and media formulations (2) Co-culture conditions for gamma/delta T cells and designer aAPC (3) Intracellular cytokine production, Luminex, and neutralizing antibody cytolysis assays (4) Lentivirus packaging and transduction of CAOV3 cells Contents of the Supplemental Data include figures: (1) An example of the gating strategy for gamma/delta T-cell analyses (2) Schematic of DNA plasmid pLVU3G-effLuc-T2A-mKateS158A used to co-express enhanced firefly luciferase (effLuc) and mKate (3) Expression of activation markers CD38 and CD95 on propagated gamma/delta T cells (4) aAPC developed for co-culture with gamma/delta T cells to determine the impact of introduced co-stimulatory molecules (5) Expansion of UCB-derived gamma/delta T cells on aAPC with IL-2 and IL-21 (6) Surface expression of TCRdelta1 and TCRdelta2 chains on gamma/delta T cells derived from PBMC prior to propagation (7) Abundance of Vdelta and Vgamma mRNA species in gamma/delta T cells prior to ex vivo numeric expansion (8) Surface expression of TCRdelta1 and TCRdelta2 chains on gamma/delta T cells derived from UCB and propagated on aAPC with IL-2 and IL-21 (9) mRNA expression of shared Valpha/Vdelta alleles in gamma/delta T cells separated and then propagated on aAPC, IL-2, and IL-21 (10) Abundance of mRNA species coding for Vgamma chains in gamma/delta T-cell subsets (11) In vitro lysis of tumor cell line panel by polyclonal gamma/delta T cells (12) Specific lysis of hematological and solid tumor cells by Vdelta T-cell subsets (13) Immunophenotypes associated with PBMC-derived Vdelta T-cell subsets expanded on aAPC/IL-2/IL-21. Supplemental Table 1 - details the antibodies used in this study, where they were purchased, and their hybridoma clone.

Purpose: Adoptive transfer of natural killer (NK) cells combined with tumor-specific monoclonal antibodies (mAb) has therapeutic potential for malignancies. We determined if large numbers of activated NK (aNK) cells can be grown ex vivo from peripheral blood mononuclear cells (PBMC) of children with high-risk neuroblastoma using artificial antigen-presenting cells (aAPC).Experimental Design: Irradiated K562-derived Clone 9.mbIL21 aAPC were cocultured with PBMC, and propagated NK cells were characterized with flow cytometry, cytotoxicity assays, Luminex multicytokine assays, and a nonobese diabetic/severe combined immunodeficient (NOD/SCID) mouse model of disseminated neuroblastoma.Results: Coculturing patient PBMC with aAPC for 14 days induced 2,363- ± 443-fold expansion of CD56+CD3−CD14− NK cells with 83% ± 3% purity (n = 10). Results were similar to PBMC from normal donors (n = 5). Expression of DNAM-1, NKG2D, FcγRIII/CD16, and CD56 increased 6- ± 3-, 10- ± 2-, 21- ± 20-, and 18- ± 3-fold, respectively, on day 14 compared with day 0, showing activation of NK cells. In vitro, aNK cells were highly cytotoxic against neuroblastoma cell lines and killing was enhanced with GD2-specific mAb ch14.18. When mediating cytotoxicity with ch14.18, release of TNF-α, granulocyte macrophage colony-stimulating factor, IFN-γ, sCD40L, CCL2/MCP-1, CXCL9/MIG, and CXCL11/I-TAC by aNK cells increased 4-, 5-, 6-, 15-, 265-, 917-, and 363-fold (151–9,121 pg/mL), respectively, compared with aNK cells alone. Survival of NOD/SCID mice bearing disseminated neuroblastoma improved when treated with thawed and immediately intravenously infused cryopreserved aNK cells compared with untreated mice and was further improved when ch14.18 was added.Conclusion: Propagation of large numbers of aNK cells that maintain potent antineuroblastoma activities when cryopreserved supports clinical testing of adoptive cell therapy with ch14.18. Clin Cancer Res; 19(8); 2132–43. ©2013 AACR.

PDF file - 1549K, Supplemental Figure 1. Cytokine and chemokine release from K562 Clone 9.mbIL21 aAPC-expanded effector cells after 24-hour incubation with neuroblastoma cell line CHLA-255-Fluc alone or with anti-GD2 antibody ch14.18. Supplemental Figure 2. Anti-tumor activity of K562 Clone 9.mbIL21 aAPC-expanded and cryopreserved effector cells from a normal donor PBMC that were thawed and cultured for 3 days or thawed and immediately injected intravenously into NOD/SCID mice beginning 7 days after they had received 106 CHLA-255-Fluc neuroblastoma cells intravenously. Supplemental Figure 3. Anti-tumor activity of dose-dense treatment with K562 Clone 9.mbIL21 aAPC-expanded and cryopreserved aNK cells derived from a patient�s PBMC. Supplemental Figure 4. Anti-tumor activity of K562 Clone 9.mbIL21 aAPC-expanded and cryopreserved aNK cells derived from a patient�s PBMC when treatment was begun 21 days (late treatment) after tumor cell injection.

PDF file - 89K, Supplemental Table 1. List of antibodies used in this study. Supplemental table 2. Cytokines/chemokines excluded from the Luminex� panels.

Supplementary Figure 1 from Reprogramming CD19-Specific T Cells with IL-21 Signaling Can Improve Adoptive Immunotherapy of B-Lineage Malignancies

Supplementary Materials and Methods

Activation and functional response of Cetux-CAR+ and Nimo-CAR+ T cells in response to cell lines with varying endogenous EGFR expression

Antibodies used in study

Engraftment of intracranial tumor and CAR+ T-cell phenotype prior to administration of T cells

Production of TNF-a by Cetux-CAR and Nimo-CAR+ T cells in response to different densities of EGFR

Ratio of CD4 and CD8 on Cetux-CAR+ and Nimo-CAR+ T cells

Surface expression of CAR during co-culture with tEGFR+ EL4 or CAR-L+ EL4

Schematic of plasmids and AaPC

Opinion statementNatural killer (NK) cells have played a critical—if largely unrecognized or ignored—role in the treatment of B cell non-Hodgkin lymphoma (NHL) since the introduction of CD20-directed immunotherapy with rituximab as a cornerstone of therapy over 25 years ago. Engagement with NK cells leading to lysis of NHL targets through antibody-dependent cellular cytotoxicity (ADCC) is a critical component of rituximab’s mechanism of action. Despite this important role, the only aspect of B cell NHL therapy that has been adopted as standard therapy that even indirectly augments or restores NK cell function is the introduction of obinutuzumab, a CD20 antibody with enhanced ability to engage with NK cells. However, over the last 5 years, adoptive immunotherapy with effector lymphocytes of B cell NHL has experienced tremendous growth, with five different CAR T cell products now licensed by the FDA, four of which target CD19 and have approved indications for some subtype of B cell NHL—axicabtagene ciloleucel, brexucabtagene autoleucel, lisocabtagene maraleucel, and tisagenlecleucel. These T cell-based immunotherapies essentially mimic the recognition, activation pathway, and cytotoxic machinery of a CD19 antibody engaging NK cells and lymphoma targets. Despite their efficacy, these T cell-based immunotherapies have been difficult to implement because they require 4–6 weeks of manufacture, are costly, and have significant toxicities. This renewed interest in the potential of cellular immunity—and the manufacturing, supply chain, and administration logistics that have been addressed with these new agents—have ignited a new wave of enthusiasm for NK cell-directed therapies in NHL. With high safety profiles and proven anti-lymphoma efficacy, one or more new NK cell-directed modalities are certain to be introduced into the standard toolbox of NHL therapy within the next few years, be it function-enhancing cytokine muteins, multi-domain NK cell engagers, or adoptive therapy with expanded or genetically modified NK cells.

Key Points AHR directly regulates a wide range of genes in NK cells, including those involved in cell signaling, oxidative stress, and metabolism.Knowing of the repertoire of genes regulated by AHR may help us better understand NK-cell dysfunction mediated by AHR ligands in cancer., Visual Abstract, The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor that regulates cellular processes in cancer and immunity, including innate immune cell development and effector function. However, the transcriptional repertoire through which AHR mediates these effects remains largely unexplored. To elucidate the transcriptional elements directly regulated by AHR in natural killer (NK) cells, we performed RNA and chromatin immunoprecipitation sequencing on NK cells exposed to AHR agonist or antagonist. We show that mature peripheral blood NK cells lack AHR, but its expression is induced by Stat3 during interleukin-21–driven activation and proliferation, coincident with increased NCAM1 (CD56) expression resulting in a CD56bright phenotype. Compared with control conditions, NK cells expanded in the presence of the AHR antagonist, StemRegenin-1, were unaffected in proliferation or cytotoxicity, had no increase in NCAM1 transcription, and maintained the CD56dim phenotype. However, it showed altered expression of 1004 genes including those strongly associated with signaling pathways. In contrast, NK cells expanded in the presence of the AHR agonist, kynurenine, showed decreased cytotoxicity and altered expression of 97 genes including those strongly associated with oxidative stress and cellular metabolism. By overlaying these differentially expressed genes with AHR chromatin binding, we identified 160 genes directly regulated by AHR, including hallmark AHR targets AHRR and CYP1B1 and known regulators of phenotype, development, metabolism, and function such as NCAM1, KIT, NQO1, and TXN. In summary, we define the AHR transcriptome in NK cells, propose a model of AHR and Stat3 coregulation, and identify potential pathways that may be targeted to overcome AHR-mediated immune suppression.

Patients with acute myeloid leukaemia (AML) have a five-year survival rate of 28·7%. Natural killer (NK)-cell have anti-leukaemic activity. Here, we report on a series of 13 patients with high-risk R/R AML, treated with repeated infusions of double-bright (CD56bright /CD16bright ) expanded NK cells at an academic centre in Brazil. NK cells from HLA-haploidentical donors were expanded using K562 feeder cells, modified to express membrane-bound interleukin-21. Patients received FLAG, after which cryopreserved NK cells were thawed and infused thrice weekly for six infusions in three dose cohorts (106 -107 cells/kg/infusion). Primary objectives were safety and feasibility. Secondary endpoints included overall response (OR) and complete response (CR) rates at 28-30 days after the first infusion. Patients received a median of five prior lines of therapy, seven with intermediate or adverse cytogenetics, three with concurrent central nervous system (CNS) leukaemia, and one with concurrent CNS mycetoma. No dose-limiting toxicities, infusion-related fever, or cytokine release syndrome were observed. An OR of 78·6% and CR of 50·0% were observed, including responses in three patients with CNS disease and clearance of a CNS mycetoma. Multiple infusions of expanded, cryopreserved NK cells were safely administered after intensive chemotherapy in high-risk patients with R/R AML and demonstrated encouraging outcomes.

High grade gliomas are identified as malignant central nervous tumors that spread rapidly and have a universally poor prognosis. Historically high grade gliomas in the pediatric population have been treated similarly to adult high grade gliomas. For the first time, the most recent classification of central nervous system tumors by World Health Organization has divided adult from pediatric type diffuse high grade gliomas, underscoring the biologic differences between these tumors in different age groups. The objective of our review is to compare high grade gliomas in the adult versus pediatric patient populations, highlighting similarities and differences in epidemiology, etiology, pathogenesis and therapeutic approaches. High grade gliomas in adults versus children have varying clinical presentations, molecular biology background, and response to chemotherapy, as well as unique molecular targets. However, increasing evidence show that they both respond to recently developed immunotherapies. This review summarizes the distinctions and commonalities between the two in disease pathogenesis and response to therapeutic interventions with a focus on immunotherapy.

SUMMARYNatural killer (NK) cells are emerging as a promising therapeutic option in cancer. To better understand how cancer cells evade NK cells, we studied interacting NK and blood cancer cells using single-cell and genome-scale functional genomics screens. At single-cell resolution, interaction of NK and cancer cells induced distinct activation states in both cell types depending on the cancer cell lineage and molecular phenotype, ranging from more sensitive myeloid to more resistant B-lymphoid cancers. CRISPR screens uncovered cancer cell-intrinsic genes driving sensitivity and resistance, including antigen presentation and death receptor signaling mediators, adhesion molecules, protein fucosylation genes, and transcriptional regulators. CRISPR screens with a single-cell transcriptomic readout revealed how these cancer cell genes influenced the gene expression landscape of both cell types, including regulation of activation states in both cancer and NK cells by IFNγ signaling. Our findings provide a resource for rational design of NK cell-based therapies in blood cancers.HIGHLIGHTSTranscriptomic states of interacting NK cells and cancer cells depend on cancer cell lineageMolecular correlates of increased sensitivity of myeloid compared to B-lymphoid cancers include activating receptor ligands NCR3LG1, PVR, and ULBP1New regulators of NK cell resistance from 12 genome-scale CRISPR screens include blood cancer-specific regulators SELPLG, SPN, and MYBSingle-cell transcriptomics CRISPR screens targeting 65 genome-wide screen hits identify MHC-I, IFNy, and NF-κB regulation as underlying mechanisms

Natural killer (NK) cells possess an innate ability to recognize cancer and are key mediators of cytotoxic efficacy for anticancer antibodies. Recent advances in the ability to generate, qualify, and safely infuse NK cells have led to a wide variety of clinical trials in oncology. Although their efficacy is best established for liquid cancers, their potential application in solid cancers has received increased attention. Here, we provide general background across a disparate group of exemplary solid tumors for which there is evidence for an NK cell role, discuss NK cell recognition motifs specific to each and murine and human studies of each that are supportive of NK cell adoptive immunotherapy, and end with special considerations relevant to the solid tumor microenvironment.

Background: Natural killer (NK) cells are emerging as a promising therapeutic option in cancer. To better understand how cancer cells evade NK cells, we studied interacting NK and blood cancer cells using single-cell and genome-scale functional genomics screens. Methods: We performed multiplexed single-cell RNA-seq (scRNA-seq) on co-cultures of NK cells and 26 cell lines representing diverse blood cancers. Using screens of pooled DNA-barcoded cell lines (PRISM), we quantified the sensitivity of over 60 blood cancer cell lines and integrated the results with CCLE multi-omics to uncover molecular correlates of tumor cell susceptibility to NK cells. We performed 12 genome-scale CRISPR loss-of-function and gain-of-function screens of cancer-cell intrinsic NK cell resistance mechanisms across 7 blood cancer cell lines. Finally, we investigated the mechanisms-of-action of 65 genome-scale screen hits using CRISPR screens with scRNA-seq readout in both tumor and NK cells. Results: At single-cell resolution, interaction of NK and cancer cells induced distinct activation states in both cell types depending on the cancer cell lineage and molecular phenotype. NK cells transitioned either into an activated state characterized by 4-1BB, GITR, TIM-3, and TIGIT or a state marked by type I interferon signature. Tumor cells responded to NK cell attack by activating interferon gamma (IFNy) signaling, inducing MHC class I. The activation states correlated with sensitivity to NK cells, ranging from more sensitive myeloid to more resistant B-lymphoid cancers. Molecular correlates of increased sensitivity included expression of activating receptor ligands NCR3LG1, PVR, and ULBP1, whereas TRAF3 mutations correlated with resistance. CRISPR screens uncovered cancer cell-intrinsic genes driving sensitivity and resistance, including antigen presentation and death receptor signaling mediators and adhesion molecules. The screens identified new ​​blood cancer-specific NK cell inhibitory regulators (SELPLG, SPN, and MYB ) and genes previously underappreciated in NK cell evasion, including protein fucosylation and transcriptional regulators (e.g. GFI1B). CRISPR screens with scRNA-seq readout identified MHC-I, IFNy, and NF-κB regulation as underlying mechanisms. Cancer cell knockout of positive regulators of NK cell response (CD58, NCR3LG1) induced an inactive NK cell state, providing experimental evidence how cancer cell-intrinsic genetic alterations can shape the molecular profile of attacking immune cells to promote immune evasion. Conclusions: By integrating diverse functional genomics screens and patient genomic profiles, we provide a comprehensive landscape of potential biomarkers and functionally validated genetic mechanisms which influence how NK cells recognize and kill malignant cells. The results offer a roadmap to facilitate development of NK-cell based immunotherapy for blood cancers and beyond. Citation Format: Olli Dufva, Sara Gandolfi, Jani Huuhtanen, Olga Dashevsky, Khalid Saeed, Jay Klievink, Petra Nygren, Jonas Bouhlal, Jenni Lahtela, Anna Näätänen, Bishwa Ghimire, Tiina Hannunen, Pekka Ellonen, Hanna Duàn, Jason Theodoropoulos, Essi Laajala, Jouni Härkönen, Petri Pölönen, Merja Heinäniemi, Shizuka Yamano, Ryosuke Shirasaki, David Barbie, Jennifer Roth, Rizwan Romee, Michal Sheffer, Harri Lähdesmäki, Dean A. Lee, Ricardo De Matos Simoes, Matti Kankainen, Constantine S. Mitsiades, Satu Mustjoki. Single-cell functional genomics of natural killer cell evasion by tumor cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 667.

Multiple myeloma (MM) is a plasma cell neoplasm that commonly expresses CD38. Daratumumab (DARA), a human monoclonal antibody targeting CD38, has significantly improved the outcome of patients with relapsed or refractory MM, but the response is transient in most cases. Putative mechanisms of suboptimal efficacy of DARA include downregulation of CD38 expression and overexpression of complement inhibitory proteins on MM target cells as well as DARA-induced depletion of CD38high natural killer (NK) cells resulting in crippled antibody-dependent cellular cytotoxicity (ADCC). Here, we tested whether maintaining NK cell function during DARA therapy could maximize DARA-mediated ADCC against MM cells and deepen the response. We used the CRISPR/Cas9 system to delete CD38 (CD38KO) in ex vivo expanded peripheral blood NK cells. These CD38KO NK cells were completely resistant to DARA-induced fratricide, showed superior persistence in immune-deficient mice pretreated with DARA, and enhanced ADCC activity against CD38-expressing MM cell lines and primary MM cells. In addition, transcriptomic and cellular metabolic analysis demonstrated that CD38KO NK cells have unique metabolic reprogramming with higher mitochondrial respiratory capacity. Finally, we evaluated the impact of exposure to all-trans retinoic acid (ATRA) on wild-type NK and CD38KO NK cell function and highlighted potential benefits and drawbacks of combining ATRA with DARA in patients with MM. Taken together, these findings provide proof of concept that adoptive immunotherapy using ex vivo expanded CD38KO NK cells has the potential to boost DARA activity in MM.

Background Optimal expansion of therapeutic natural killer (NK) cell products has required media supplementation with human or fetal bovine serum, which raises safety and regulatory concerns for clinical manufacturing. Serum-free media (SFM) have been optimized for T-cell expansion, but few SFM systems have been developed for NK cells. Here, we compare six commercial clinical-grade SFM with our standard fetal bovine serum–containing medium for their ability to support NK cell expansion and function. Methods Human peripheral blood NK cells were expanded in selected media by recursive weekly stimulation with K562-based feeder cells expressing membrane-bound interleukin-21 and CD137L. Expansion was the primary readout, and the best-performing SFM was then compared with standard medium for cytotoxicity, phenotype, degranulation and cytokine secretion. Multiple lots were compared for consistency, and media was analyzed throughout for nutrient consumption and metabolic byproducts. Results TexMACS, OpTmizer, SCGM, ABS-001 and StemXVivo demonstrated equal or inferior NK cell expansion kinetics compared with standard medium, but expansion was markedly superior with AIM V + 5% Immune Cell Serum Replacement (ICSR; mean 5448 vs. 2621-fold expansion in 14 days). Surprisingly, NK cells expanded in AIM V + ICSR also showed increased cytotoxicity, tumor necrosis factor α secretion and DNAM-1, NKG2D, NKp30, FasL, granzyme B and perforin expression. Lot-to-lot variability was minimal. Glucose and glutamine consumption were inversely related to lactate and ammonia production. Discussion The AIM V + ICSR SFM system supports excellent ex vivo expansion of clinical-grade NK cells with the phenotype and function needed for adoptive immunotherapy.

Clinical response rates after adoptive cell therapy (ACT) are highly correlated with in vivo persistence of the infused T cells. However, antigen-specific T cells found in tumor sites are often well-differentiated effector cells with limited persistence. Central memory CD8+ T cells, capable of self-renewal, represent desirable ACT products. We report here that exposure to a histone deacetylase inhibitor (HDACi) and IL21 could reprogram differentiated human CD8+ T cells into central memory–like T cells. Dedifferentiation of CD8+ T cells was initiated by increased H3 acetylation and chromatin accessibility at the CD28 promoter region. This led to IL21-mediated pSTAT3 binding to the CD28 region, and subsequent upregulation of surface CD28 and CD62L (markers of central memory T cells). The reprogrammed cells exhibited enhanced proliferation in response to both IL2 and IL15, and a stable memory-associated transcriptional signature (increased Lef1 and Tcf7). Our findings support the application of IL21 and HDACi for the in vitro generation of highly persistent T-cell populations that can augment the efficacy of adoptively transferred T cells.