A Multi-Omics Approach for Evaluating the Impact of Cytokines and Donor Source on NK Cell Expansion

Abstract Introduction As the field of cancer immunotherapy progresses, natural killer (NK) cells have become an ideal candidate for targeting multiple types of cancer. These cells are able to selectively target virally infected cells and tumor cells without damaging the healthy cells of the immune system making them an ideal candidate for treatment in AML, CML, MM, Neuroblastoma, Breast Cancer, and Renal Cell Carcinoma. While NK cells have shown their potential as immunotherapy agents, one of the biggest challenges includes expanding and harvesting enough cells for multiple transfusions when there are limited numbers of starting cells. Our group developed an NK cell expansion platform utilizing membrane bound-IL21 (mbIL-21), which allows for sustained proliferation in order to generate high numbers of cells. Continued research supported that mbIL-21 enables sustained NK cell proliferation compared to mbIL-15 due to mbIL-21 activation through STAT3 signaling. Recent shifts to umbilical cord blood have led to research using cord blood (CB) derived NK cells. However, this presents a challenge as a viable clinical option as others have shown that the expansion of these CB-derived NK cells have been shown to produce less potent NK cells. Here, we will use feeder cells expressing either mbIL15 or mbIL21 to expand NK cells (as is currently done in clinical trials), to deeply compare and contrast the impact of these two cytokines on NK cell biology and simultaneously compare and contrast the use of peripheral-derived NK cells and CB-derived NK cells. Methods NK cells were obtained from healthy donor leukopacks (n =4) and isolated using RosetteSep human NK cell enrichment cocktail. These samples were stimulated with either mbIL-21 or mbIL-15 irradiated feeder cells (IFC) and cultured in AIM-V media supplemented with 100 I/U of IL-2 and immune cell serum replacement (ICSR). Cord Blood NK cells were obtained from OrganaBio (n=4) and stimulated with mbIL-21 IFC . The cells were cultured in AIM-V media supplemented with 100 I/U of IL-2 and ICSR (complete media). All NK cell expansions were stimulated at day 0 and day 7, as previously described, and cell cultures were assessed every 2-3 days keep cells at an optimal concentration by supplementing with complete media. Cells were collected at day 14 to capture a picture before mbIL-15 expanded cells became senescent and were immediately used for cytokine production assays, RNA collected, ATAC-seq collection, and CyTOF fixation. The remaining cells were frozen for cytotoxicity assays, mitochondrial function assays, and degranulation assays. For mitochondrial function and cytotoxicity assays, cells were thawed and cultured in complete media for two days before use. Results Overall, the peripheral blood NK cells expanded with mbIL21 showed an increase in expansion at day 14 when compared to those expanded with mbIL15. NK cells expanded with mbIL21 demonstrated a more consistent cytotoxicity profile against CHLA-136, a neuroblastoma cell line, compared to mbIL21 expanded NK cells when cultured at a 0.5:1 ratio (E:T). While the results were not statistically significant at 14 days, previous data demonstrates that we see increased changes at 21 days. Of note, we had an overall more reproducible serial killing ability in the IL21 expanded samples compared to the variability observed in IL15-expanded samples. We saw a significant difference (p < 0.0001) in peripheral blood NK cells expanded with mbIL21 compared to cb-NK cells expanded with mbIL21 with a 0.5:1 ratio (E:T) against CHLA136. mbIL-21 expanded peripheral blood NK cells had a higher maximum respiration capacity than those expanded with mbIL-15 when measured under mitochondrial stress via seahorse assay (p= 0.016). Conclusions Our data showed that the cells began to show a different phenotypic profile at day 14 depending on the type of NK cell and the type of cytokine used for stimulation. By day 14 data supported that mbIL-21 expanded cells can better survive under low glucose conditions such as tumor microenvironments than mbIL15 expanded cells. The cytotoxicity data demonstrated that peripheral derived NK cells have improved serial killing ability than the cb-NK cells and we will continue to test their ability against additional tumor cell lines that have various HLA-typing. To further elucidate these differences, we will use our RNA-seq, ATAC-seq, and CyTOF data to understand what is causing the different profiles. Figure 1 Figure 1. Disclosures Lee: Kiadis Pharma: Divested equity in a private or publicly-traded company in the past 24 months, Honoraria, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding; Courier Therapeutics: Current holder of individual stocks in a privately-held company.

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Directed Differentiation of Mobilized Hematopoietic Stem and Progenitor Cells into Functional NK Cells with Enhanced Antitumor Activity

Cells ◽

10.3390/cells9040811 ◽

2020 ◽

Vol 9 (4) ◽

pp. 811

Author(s):

Pranav Oberoi ◽

Kathrina Kamenjarin ◽

Jose Francisco Villena Ossa ◽

Barbara Uherek ◽

Halvard Bönig ◽

...

Keyword(s):

Nk Cells ◽

Progenitor Cells ◽

Peripheral Blood ◽

Cell Expansion ◽

Nk Cell ◽

Ex Vivo ◽

Feeder Cells ◽

Hematopoietic Stem ◽

Cd34 Cells ◽

Stem And Progenitor Cells

Obtaining sufficient numbers of functional natural killer (NK) cells is crucial for the success of NK-cell-based adoptive immunotherapies. While expansion from peripheral blood (PB) is the current method of choice, ex vivo generation of NK cells from hematopoietic stem and progenitor cells (HSCs) may constitute an attractive alternative. Thereby, HSCs mobilized into peripheral blood (PB-CD34+) represent a valuable starting material, but the rather poor and donor-dependent differentiation of isolated PB-CD34+ cells into NK cells observed in earlier studies still represents a major hurdle. Here, we report a refined approach based on ex vivo culture of PB-CD34+ cells with optimized cytokine cocktails that reliably generates functionally mature NK cells, as assessed by analyzing NK-cell-associated surface markers and cytotoxicity. To further enhance NK cell expansion, we generated K562 feeder cells co-expressing 4-1BB ligand and membrane-anchored IL-15 and IL-21. Co-culture of PB-derived NK cells and NK cells that were ex-vivo-differentiated from HSCs with these feeder cells dramatically improved NK cell expansion, and fully compensated for donor-to-donor variability observed during only cytokine-based propagation. Our findings suggest mobilized PB-CD34+ cells expanded and differentiated according to this two-step protocol as a promising source for the generation of allogeneic NK cells for adoptive cancer immunotherapy.

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CD16+CD56− NK Cells in the Peripheral Blood of Cord Blood Transplant Recipients: A Unique Subset of Immature NK Cells Possibly Associated with Graft-Versus-Leukemia Effect.

Blood ◽

10.1182/blood.v110.11.1074.1074 ◽

2007 ◽

Vol 110 (11) ◽

pp. 1074-1074 ◽

Cited By ~ 1

Author(s):

Xuzhang Lu ◽

Yokio Kondo ◽

Hiroyuki Takamatsu ◽

Hiroshita Yamazaki ◽

Zhirong Qi ◽

...

Keyword(s):

Stem Cell ◽

Stem Cell Transplantation ◽

Cord Blood ◽

Nk Cells ◽

Cell Transplantation ◽

Peripheral Blood ◽

Nk Cell ◽

Flow Cytometric Analysis ◽

Cell Subset ◽

Leukemic Cells

Abstract NK cells play a major role in the activity of graft-versus-host (GVL) effect after an HLA-mismatched stem cell transplantation. In unrelated cord blood transplantation (CBT) where there is often an HLA mismatch between the donor and recipient, NK cells may also play a vital role, though their roles have not been extensively studied. Cord blood (CB) is known to have a unique subset of NK cells characterized by a CD16+CD56− phenotype. CD16+CD56− NK cells in CB are thought to be progenitors of CD16+CD56+ NK cells because CD16+CD56− NK cells acquires CD56 expression after in vitro culture in the presence of IL-2. However, the function of this immature NK cell subset after CBT remains unknown. A marked increase in the number of CD16+CD56- NK cells in the peripheral blood of an HLA-mismatched CBT recipient with acute myeloid leukemia (AML) was recently observed. A 56-year old male, who received a reduced intensity CBT following a full relapse after allogeneic stem cell transplantation from an HLA-matched sibling donor, showed an increase in the copy number of WT-1 mRNA in the peripheral blood around day 80 after the CBT, but the WT-1 copy number decreased from 1500/microliter RNA to 230/microliter RNA in association with the increase in the number of CD16+CD56- NK cells, and his molecular remission lasted more than 1.5 years thereafter. This case prompted an investigation of CD16+CD56− NK cells in the peripheral blood after allogeneic stem cell transplantation. A similar increase in the proportion of CD16+CD56− NK cells (20% or more) in the peripheral blood CD16+ NK cells was observed in 64% (7/11) of CBT recipients, all of whom maintained remission, but in none of the 11 bone marrow and 8 peripheral blood stem cell transplant recipients examined (Figure 1). CD16+CD56− NK cells in CBT recipients expressed receptors specific to NK cells such as NKp30 and NKp46 same level as CD16+CD56− NK cells of fresh CB cells. CD16+CD56− NK cells isolated from CBT recipients became CD56+ when they were cultured in the presence of IL-2 with or without K562-mb15-4-1BBL. When cultured NK cells derived from the CD16+CD56− NK cells were separated into CD158b+ and CD158b− cells, CD158b+ cells failed to kill 721–221 cells transfected with HLA-C*0301 while they killed untransfected or HLA-C*0401-transfected 221 cells. Despite the presence of the corresponding KIR ligand (C*0304), cultured CD16+CD56− NK cells showed cytotoxicity against the patient’s leukemic cells. These findings suggest that an increase in the proportion of CD16+CD56− NK cells is unique to recipients of CBT and that this immature NK-cell subset in CBT recipients may undergo differentiation into mature NK cells in vivo capable of killing residual leukemic cells, thereby contributing to the GVL effect regardless of the presence of the KIR ligand. Figure 1 Flow cytometric analysis of CD3-CD16+CD56-cells in peripheral blood of SCT recipients and healthy individual.Examples of three-flourescence cytofluorometric analysis of fresh isolated PBMC stained with CD3,CD56 and CD16 in different SCT patients and health individuals. The characterization of the unusual CD56-CD16+ cell subset expend only in the CBT individual(a). Presenting cellware gated on CD3-cells Figure 1. Flow cytometric analysis of CD3-CD16+CD56-cells in peripheral blood of SCT recipients and healthy individual.Examples of three-flourescence cytofluorometric analysis of fresh isolated PBMC stained with CD3,CD56 and CD16 in different SCT patients and health individuals. The characterization of the unusual CD56-CD16+ cell subset expend only in the CBT individual(a). Presenting cellware gated on CD3-cells

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Differential Genomic and Proteomic Signatures In Cord Blood (CB) Vs Peripheral Blood (PB) CD56+Dim NK Cells: Over Expression of CD34 In CB Vs PB CD56+Dim NK Cells

Blood ◽

10.1182/blood.v116.21.2781.2781 ◽

2010 ◽

Vol 116 (21) ◽

pp. 2781-2781

Author(s):

Nancy S Day ◽

Evan Shereck ◽

Janet Ayello ◽

Catherine McGuinn ◽

Prakash Satwani ◽

...

Keyword(s):

Cell Death ◽

Programmed Cell Death ◽

Cord Blood ◽

Nk Cells ◽

Peripheral Blood ◽

Nk Cell ◽

Conflicts Of Interest ◽

Hematopoietic Stem ◽

Over Expression ◽

Apoptotic Genes

Abstract Abstract 2781 Background. Umbilical cord blood (UCB) is a viable alternative source of allogeneic hematopoietic stem cells for the treatment of both malignant and non-malignant disease (Cairo et al BBMT 2008). UCB transplantation (UCBT) is known to be associated with decrease severe acute graft-versus-host disease (GvHD) compared to unrelated bone marrow (BM) and peripheral blood (PB) transplantation; however, it is associated with delayed hematopoietic and immune reconstitution (Szabolcs/Cairo et al Seminars in Hematology 2010). NK cells play important roles in both innate and adaptive immunity and are characterized as a CD56+ cell population. NK cell recovery is prompt by 2 months after hematopoietic stem cell transplantation (HSCT), while T-cell (after at least 9 mo HSCT) and B-cell (after 3 to 4 mo HSCT) reconstitutions are gradual and delayed. CD56+dim cells are primarily cytotoxic and make up 90% of PB NK populations (Shereck/Cairo PBC 2007). We previously demonstrated the ability to ex-vivo expand CB MNC into various phenotypes of CD56+dim and CD56+bright NK cells (totally 60%) and NKT cells (40%) with profound in vitro and in vivo cytotoxicity against hematological malignancies (Ayello/Cairo BBMT 2006 & Exp. Hematology 2009). Proteomic studies from our group demonstrated differential protein expression including ↑NKG2A, ↓IP3R type 3, ↓MAPKAPK5, and ↑NOTCH 2 in CB vs PB CD56+dim (Shereck/Cairo, ASH 2007; Shereck/Day/Cairo, ASBMT 2009). Objective. In these studies, we sought to determine the similarity or differences in genetic signatures in CB vs APB CD56+dim NK cells. Methods. CB MNCs were isolated on a ficoll gradient and NK CD56+16+dim cells isolated using a 2-step magnetic activated cell separation (MACS) process via a standard kit (Miltenyi Biotec). Enrichment was at least 94%. Isolated RNA from CB and PB CD56+dim cells were subjected to microarray studies (Affymetrix, U133A_2) as we have previously described (Jiang/Cairo et al J Immunol 2004). Data were analyzed by Agilent GeneSpring and Ingenuity pathway analyses. Welch test were used to perform statistical analysis and fold change of < 1.5 and values of p<0.05 were considered to be significant. Two-color ECL Plex fluorescence Western blotting (WB) was preformed to validate the proteomic data. Protein samples were separated using SDS-PAGE followed by transblotting. WB membranes were then incubated with target and control (GAPDH) primary antibodies. After rinse and wash, the membranes were further incubated with CY5 and CY3 conjugated secondary antibodies. The membranes were scanned with TYPHOON by green (532 laser and 580 filter) and red (633 laser and 670 filter) setting for CY3 and CY5, respectively, and then observed and quantified using ImageQuant. Results. CB vs PB CD56+dim cells significantly altered expressed 796 genes, in which 486 genes were over expressed, at the genomic level including: pro-apoptotic genes: CASP10 (3.1F), TNFSF11 (4.7F), CDC2 (3.0F), BCL2L1 (4.3F), NOTCH2 (1.5F); and cell development: PBX1 (7.6F), IL1RN (5.1F), CD24 (5.3F), CD34 (3.5F), CD55 (2.1F), CCL13 (2.2F). Conversely, there was significant under expression of NF1 (5.1F), MAP2K3 (1.7F), PIK3CD (2.1F), BAX (2.9F), and JUN (2.2F). Our WB results indicate that NOTCH2 (2.4F) and PBX1 (2.2F) proteins are increased in CB vs PB CD56+dim NK cells, consistent with our proteomic results. Conclusion. These results suggest that CB vs PB CD56+dim NK are more prone to undergo programmed cell death (apoptosis) secondary to over expression of numerous pro-apoptotic genes, and may be earlier in development (pro-NK) with over expression of the CD34 gene. Furthermore, decrease CB vs PB NK cytotoxicity maybe in part secondary to increase programmed cell death in particularly increase NOTCH2 at the genomic and proteomic levels. (The first two authors contribute equally.) Disclosures: No relevant conflicts of interest to declare.

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T/NK Lymphocytosis in CML Ph+ Patients During Dasatinib Therapy.

Blood ◽

10.1182/blood.v114.22.3279.3279 ◽

2009 ◽

Vol 114 (22) ◽

pp. 3279-3279

Author(s):

Antonella Russo Rossi ◽

Alessandra Ricco ◽

Paola Carluccio ◽

Mario Delia ◽

Manuela Leo ◽

...

Keyword(s):

Nk Cells ◽

Peripheral Blood ◽

Nk Cell ◽

Conflicts Of Interest ◽

Cytotoxic T Cell ◽

Molecular Response ◽

Imatinib Treatment ◽

Long Term Outcome ◽

Significant Difference ◽

The Impact

Abstract Abstract 3279 Poster Board III-1 Background. Some authors reported that Natural Killer (NK) cells from CML patients are defective in NK cell activity and NK cell number decrease as the disease progresses to the advanced phase and probably the abnormal BCR/ABL gene causes abnormal NK cell differentiation.TK inhibitors reduce BCR/ABL transcription and could restore NK cell numbers and/or function.Moreover Dasatinib,by the blockade of SRC Kinases,could affect the development of NK cells as well as T-lymphocytes.Our aim was to verify the impact of Dasatinib treatment on T CD8+ and NK cells modulation. Methods. We evaluated 24 patients with CML resistant/intollerant to Imatinib and treated with Dasatinib at a starting dose of 70 mg/BID or 100 mg/QD.Blood count were monitored; lymphocytosis has been definited by an increased number of peripheral blood lymphocyte counts ≥ 3.0×10(e)9/L and by the predominance of LGLs in peripheral blood smear. Immunophenotyping was done with flow-cytometry using antibodies against the following antigens: CD2, CD3, CD4, CD5, CD7, CD8, CD16, and CD56. Results. With a median of 19 mo. of Dasatinib therapy (range 3–43), 15/24 cases (62.5%) developed peripheral blood lymphocytosis. Median onset of lymphocytosis was 3 months after the initiation of Dasatinib therapy (range 1–12) and duration was 14 months (range 6–40).Lymphocytosis was CD3+/CD8+/Cytotoxic T Cell in 9 patients (60%) and CD3-/CD16+/CD56+/NK Cell in 6 patients (40%).In all 15 patients no symptoms or signs suggestive of LGL leukemia or viral infections were documented.There was no significant difference in terms of the frequency of severe adverse events, including pleural effusion between patients with and without lymphocytosis. 11(73%) of the 15 patients who developed lymphocytosis achieved MMolR and 4/11 presented Bcr/Abl mutation at the time of imatinib treatment (F317L, E255K, F359V, E255K),whereas only 3(33%) of the 9 patients without lymphocytosis achieved MMolR and 1/3 presented Bcr/Abl mutation (F359V). Moreover molecular response was earlier in the group of patients with lymphocytosis (8vs12 mo.). Conclusions. The development of lymphocytosis in our patients seems to be associated to an improved response to dasatinib in terms of molecular response and time to response. The assessment of higher frequency lymphocytosis requires further analysis; a larger patients'cohort should be needed to explore the biological role of lymphocytosis and the impact on the long term outcome in patients treated with dasatinib. Disclosures: No relevant conflicts of interest to declare.

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Generation Of Clinical Grade, Highly Cytotoxic NK Cells Using Feeder Cell Free, Particle-Based Technology

Blood ◽

10.1182/blood.v122.21.4503.4503 ◽

2013 ◽

Vol 122 (21) ◽

pp. 4503-4503

Author(s):

Jerremiah Oyer ◽

Igarashi Y Robert ◽

Colosimo Dominic ◽

Melhem M. Solh ◽

Yasser Khaled ◽

...

Keyword(s):

Nk Cells ◽

Cell Lines ◽

Peripheral Blood ◽

Nk Cell ◽

Ex Vivo ◽

Free Particle ◽

Leukemia Cell ◽

High Dose ◽

Feeder Cells ◽

Peripheral Blood Mononuclear

NK cell immunotherapy shows exciting promise, but inconsistency and variability remain as a significant challenge. Since NK cells comprise a small fraction (∼5%) of the peripheral blood mononuclear cell fraction, expansion of NK cells in vivo or ex vivo is a critical requirement to attain therapeutically effective dosages and to observe consistent positive clinical outcomes. Most of currently developed ex vivo expansion protocols depend on co-culture with various engineered and/or cancer derived stimulator/feeder cells to induce the proliferation of NK cells. The use of accessory cells poses significant challenges to clinical transfer. Our laboratory has developed a nanoparticle-based expansion technology that utilizes particles, few hundred nanometers in size, derived from the plasma membrane (PM) of K562 feeder cells expressing IL-15 and 41BBL on their surface (PM-mb15-41BBL). These particles in combination with low concentration of IL-2 induce selective and efficient expansion of NK cells within human peripheral blood mononuclear cells (PBMC). When PBMC are stimulated with PM-mb15-41BBL over 21 days the NK cell numbers increase exponentially between days 6 and 18 of culture. The numbers of NK cell increased on average 200 fold (range 104-557, n=11, 4 donors) after 12-13 days of culture in the presence of PM-mb15-41BBL particles (at 200 µg of membrane protein/mL). The expansions with the PM particles are comparable to those in the presence of live feeder cells that gave ∼200 fold (79-895, n=11, 4 donors). The PM-particle based NK expansion is far better in comparison to NK stimulation with soluble purified 41BBL, IL-15 and IL-2, at matching concentrations, that yielded only 3 fold (1-4, n=6, 3 donors) increase in NK cells. Furthermore, the NK cells expand selectively under these conditions where they initially consisted only about 10% of the population of PBMC isolated from fresh peripheral blood, but increased to more than 95% of the cell suspension after 14 days in culture. The extent of expansion and NK cell content on day 12 of culture was dependent on the concentration of PM particles used with 200 µg of PM protein/mL being the optimal dose. Thus, PM nanoparticles can expand NK cells as efficiently and selectively as feeder cells. Furthermore, the PM-particle based expansion is more reproducible between trials and with different donors as compared to NK cell expansion induced with feeder cells (coefficient of variation 63% vs. 88%, respectively). The NK cells expanded in presence of PM-particles were highly cytotoxic against several leukemia cell lines and also against patient derived AML blasts. Expanded NK cells were 4 to 9 times more potent against AML cell lines K562, KG1 and HL-60 as compared to freshly isolated NK cells that were pre-activated with a high dose of IL-2. The PM-particle expanded NK cells also were selectively cytotoxic where they efficiently killed patient derived CD34+ leukemia blasts while sparing healthy CD34- peripheral blood cells. The expanded NK cells were observed to have an increase in the expression of major activating receptors such as NKG2D, NKp44, NKp30 and of the death receptor ligand FasL. This expression difference corresponds well with the activated cytotoxic phenotype and is likely responsible for their increased cytotoxicity against AML cells. Pilot trials in NSG mice are currently ongoing. Disclosures: Solh: Celgene: Speakers Bureau.

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147 CD155 blockade boosts alloreactive natural killer cell antitumor effects against osteosarcoma

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2020-sitc2020.0147 ◽

2020 ◽

Vol 8 (Suppl 3) ◽

pp. A160-A160

Author(s):

Monica Cho ◽

Madison Phillips ◽

Longzhen Song ◽

Amy Erbe ◽

Christian Capitini

Keyword(s):

Bone Marrow ◽

Nk Cells ◽

Natural Killer ◽

Cancer Biology ◽

Nk Cell ◽

Allogeneic Bone ◽

Cytotoxicity Assays ◽

Allogeneic Bmt ◽

The Impact

BackgroundPediatric patients with relapsed and refractory osteosarcoma have poor prognoses with few treatment options. Allogeneic bone marrow transplant (BMT) has not yet shown a graft-versus-tumor (GVT) effect for osteosarcoma. Natural killer (NK) cells demonstrate antitumor activity against osteosarcoma, but adoptively transferred NK cells have limited proliferation, cytotoxicity, and persistence in vivo. To enhance an NK-specific GVT effect, we propose blocking the poliovirus receptor CD155 checkpoint molecule, which is overexpressed on osteosarcoma and can engage both activating and inhibitory receptors on NK cells. The impact of CD155 blockade on GVT and graft-versus-host-disease (GVHD) is unknown.MethodsNK cells from C57BL/6 (B6) mice were expanded with recombinant IL-15/IL-15R and analyzed by flow cytometry. Cytotoxicity assays were performed with IL-15 expanded B6 NK cells and mKate2-expressing K7M2 murine osteosarcoma at a 1:1 ratio with blockade of CD155 and CD155 ligands. To test efficacy of NK cell infusion and CD155 blockade after allogeneic BMT, BALB/c mice were lethally irradiated, transplanted with allogeneic B6 bone marrow, and challenged with luciferase-expressing K7M2 on day 0. At day 7, mice received IL-15 expanded B6 NK cells intravenously with either anti-IgG control or anti-CD155 antibody intraperitoneally and IL-2 subcutaneously on days 7 and 11. Mice were monitored for tumor growth by bioluminescence, and toxicity by GVHD using weight loss and clinical scores.ResultsCompared to unexpanded murine NK cells, IL-15 expanded NK cells (n = 6) show increased expression of NKG2D (65.33 ± 10.77% NKG2D+, p = 0.0077; 1030 ± 177.0 MFI, p = 0.0101) and an increased ratio of the CD155 activating (CD226) to inhibitory (TIGIT) ligand expression (11.71 ± 4.121, p = 0.0362). In cytotoxicity assays with IL-15 expanded allogeneic murine NK cells (n = 3 replicates), CD155 blockade enhances K7M2 osteosarcoma lysis (60.62 ± 3.19%, p = 0.0189) compared to IgG control (29.01 ± 7.66%). CD226 blockade decreased tumor killing (10.62 ± 8.51%, p = 0.0053) compared to CD155 blockade. In vivo allogeneic murine NK cell infusion and anti-CD155 antibody treatment after allogeneic BMT decreased tumor area under the curve by 44.3% compared to IgG control, without exacerbating GVHD.ConclusionsThese findings demonstrate that blockade of CD155 enhances an allogeneic NK cell-specific GVT effect for osteosarcoma treatment without exacerbating GVHD. CD155 blockade has the potential to improve usage of allogeneic BMT and NK cell adoptive immunotherapy as a combination treatment for osteosarcoma, and perhaps other pediatric sarcomas.AcknowledgementsThis work was supported by grants from the National Institute of General Medical Sciences/NIH T32 GM008692 and Training in Cancer Biology Training Grant NIH T32 CA009135 (to MMC), St. Baldrick’s Stand up to Cancer (SU2C) Pediatric Dream Team Translational Research Grant SU2C-AACR-DT-27-17, NCI/NIH R01 CA215461, American Cancer Society Research Scholar Grant RSG- 18-104-01-LIB, and the Midwest Athletes Against Childhood Cancer (MACC) Fund (to CMC). SU2C is a division of the Entertainment Industry Foundation. Research grants are administered by the American Association for Cancer Research, the scientific partner of SU2C. The contents of this article do not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the US government.

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CD38 Knockout Primary NK Cells to Prevent "Fratricide" and Boost Daratumumab Activity

Blood ◽

10.1182/blood-2019-129456 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 870-870

Author(s):

Yuya Nagai ◽

Meisam Naeimi Kararoudi ◽

Ezgi Elmas ◽

Marcelo Pereira ◽

Syed Abbas Ali ◽

...

Keyword(s):

Nk Cells ◽

Therapeutic Effect ◽

Cell Lines ◽

Peripheral Blood ◽

Research Funding ◽

Nk Cell ◽

Sequencing Analysis ◽

Proof Of Concept ◽

Cd38 Expression ◽

The Impact

Multiple myeloma (MM) is a plasma cell neoplasm typically characterized by high and uniform CD38 expression. Daratumumab (DARA), a humanized monoclonal antibody targeting CD38 has dramatically improved the outcome of patients with refractory MM, but relapse is inevitable in most cases. DARA eliminates MM cells through several mechanisms including antibody-dependent cellular cytotoxicity (ADCC), complement-dependent cytotoxicity, and antibody-dependent cellular phagocytosis. Mechanisms of resistance to DARA include downregulated CD38 expression on target MM cells, impaired complement directed cytotoxicity via upregulation of complement inhibitory proteins (CD55, CD59), and impaired ADCC via DARA-induced NK cell "fratricide". As it relates to "fratricide", restoring ADCC by DARA-resistant NK cells has been proposed to bolster the therapeutic effect of DARA. Towards this goal, iPSC-derived CD38 knockout (CD38KO) NK cells have shown proof-of-concept for this approach but present major challenges towards clinical translation. Here, we used Cas9-RNP to generate CD38KO primary NK cells and characterized their resistance to DARA-induced "fratricide" and restoration of ADCC, but also assessed the impact of CD38 deletion on NK cell signaling and metabolism. First, primary NK cells were enriched from peripheral blood of healthy donors and expanded on feeder cells expressing mbIL-21 and 4-1BBL (PLoS One. 2012;7(1):e30264). Cas9/gRNA complexes targeting CD38 were electroporated into NK cells (J Vis Exp. 2018 Jun 14;(136)). Flow cytometric analysis revealed CD38KO efficiency was 81.9±6.9% (mean±SD, n=5). CD38KO cells were further purified to ≥ 95% purity using magnetic cell separation system. Whole genome sequencing identified no off-target mutations. CD38KO-NK cells alone showed no conjugation in the presence of DARA (Figure 1A) and no DARA-induced fratricide (Figure 1B), in stark contrast with wild type NK (WT-NK) cells. Consistent with these results, treatment of NSG mice with DARA had no impact on persistence of CD38KO-NK cells, whereas WT-NK cells were completely eliminated (NK cell frequency in peripheral blood 7 days after inoculation; WT-NK NO DARA 12.11±5.15%, KO-NK NO Dara 17.05±0.98%, WT-NK + Dara 0.21±0.08%, KO-NK + Dara 16.85±3.61%, mean±SD, n=4, p<0.01, ANOVA) (Figure 1C). To examine the functional status of CD38KO-NK cells, we used 6 MM cell lines with various CD38 expression levels (LP-1, RPMI8226, H929, MM.1s, OPM-2, and KMS-11) as well as three CD38+CD138+ primary MM samples isolated from bone marrow of newly diagnosed or relapsed patients. CD38KO-NK cells exhibited enhanced ADCC activity compared to WT-NK cells across all cell lines and primary samples tested in 4h and 24h cytotoxicity assay (Figure 1D). This was even observed in MM cells that express low levels of CD38, suggesting their potential efficacy in patients with relapsed disease after DARA (ADCC at E/T 5:1; MM.1s 7.8±1.8%(WT) vs 21.5±0.4%(KO), KMS-11 -3.5±0.6%(WT) vs 11.1±0.4%(KO), mean±SD). The CD38KO-NK cells preserved similar expansion potency as WT-NK counter parts. Next, to reveal any effect of manipulating CD38 expression on NK cells cytotoxicity and metabolism, we performed RNA sequencing analysis, which showed higher expression of several cytotoxic genes such as IFNG and GzmB in CD38-KO NK cells, and higher expression of glucose transporter genes and GAPDH, suggesting a favorable metabolic profile of these cells. Consistent with these data, we observed that CD38KO-NK cells have elevated capacity to take up glucose as measured by 2-NBDG glucose uptake assay (p=0.02, student t test). Taken together, these findings provide the proof-of-concept that CD38KO-NK cells generated from primary NK cells augment therapeutic effect of DARA and could be promising for adoptive immune cellular therapy targeting MM. Y.N. and M.N.K. contributed equally to this study. D.A.L. and G.G. contributed equally to this study. Figure 1 Disclosures Ali: Celgene: Research Funding; Poseida: Research Funding. Lee:Kiadis Pharma: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding.

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A Highly Efficient Method to Expand CD3-CD56+ NK Cells from Cord Blood Segments

Blood ◽

10.1182/blood.v112.11.3902.3902 ◽

2008 ◽

Vol 112 (11) ◽

pp. 3902-3902

Author(s):

Sumithira Vasu ◽

Maria Berg ◽

Andreas Lundqvist ◽

Muthalagu Ramanathan ◽

Rebecca Lopez ◽

...

Keyword(s):

Cord Blood ◽

Nk Cells ◽

Nk Cell ◽

K562 Cells ◽

Surface Expression ◽

Feeder Cells ◽

Cell Numbers ◽

In Vitro Expansion ◽

Cytotoxic Function

Abstract Background: The infusion of in-vitro expanded cord blood derived CD3−CD56+ NK cells could potentially be used to enhance graft-vs-tumor effects following cord blood transplantation. In order to infuse NK cells derived from the same cord unit used during allogeneic transplantation, we sought to develop a highly efficient culture method to expand large numbers of NK cells in vitro from < 1 ml of cord blood. Methods: Immunomagnetic beads were used to deplete CD3+ T-cells from thawed cord blood. CD3 depleted mononuclear cells (<0.1% CD56+) were co-cultured with either irradiated EBV-LCL feeder cells or 41BB transduced K562 cells in X-VIVO 20, 10% human AB serum, and 500 IU/ml hrIL-2 for up to 47 days. Results: Day 12 EBV-LCL expanded NK cell cultures contained up to 90% CD3−CD56+ NK cells with less than 0.5% CD3+CD56+ cells. Expanded cord blood derived CD56+NK cells had similar expression of CD16, NKG2D, LFA-1, perforin, and granzymes A and B and had similar cytotoxic function as NK cells expanded from adult PBMC. Surface expression of NK cell TRAIL increased dramatically with in vitro expansion. By day 12, TRAIL surface expression by FACS was at similar levels observed on expanded adult NK cells, although expression gradually declined with prolonged cell culture; on days 12, 20, and 34, 89%, 57% and 11% of cord derived NK cells expressed TRAIL respectively. NK cell cultures expanded with 41BB-transduced APCs had a similar phenotype and cytotoxic function against K562 cells and renal cell carcinoma (RCC) cells as EBV-LCL expanded cells. Furthermore, NK cell cytotoxicity against RCC tumor targets treated with 10 nM bortezomib for 18 hrs (bortezomib upregulates RCC surface expression of DR5) was higher than untreated RCC cells confirming the functional cytolytic activity of TRAIL expressed on cord blood derived NK cells. We next evaluated the feasibility of expanding CD56+ NK cells from thawed segments attached to the umbilical cord blood units. TNC numbers of each segment ranged from 3–9 × 106 cells. Unmanipulated thawed segments were co-cultured with irradiated EBV-LCL feeder cells as described above. On Day 12, 43% of viable cells were CD3+, 21.5% were CD3+CD56+ and 36% were CD3− CD56+. CD3−CD56+ NK cells from thawed segments increased 200 – 300 fold with in vitro expansion when maintained in culture for 2–3 weeks (Table). To enrich for pure NK cell populations, subsequent expansions were performed on CD3 depleted cells pooled from three thawed segments stimulated with EBV-LCL; cultures on day 12 contained a pure population of CD3−CD56+ NK cells with virtually an identical phenotypic and cytotoxicity profile as NK cells expanded from larger aliquots taken directly from the thawed cord blood unit. By day 33, CD3−CD56+ NK cells expanded by up to 30,000 fold; in one experiment, 391 ×106 CD3-CD56+ NK cells were expanded from only 13,000 NK cells obtained from a pool of three thawed cord segments. Conclusions: In vitro-expansion of a pure population of CD3−CD56+ cells derived from cord blood can be achieved using EBV-LCL or 41BB transduced feeder cells. Expanded cells have increased NKG2D and TRAIL expression and enhanced TRAIL-mediated tumor cytotoxicity. Even with very low starting numbers of TNCs, substantial numbers of CD3+, CD3+56+ and CD3−CD56+ cells can be expanded in vitro from thawed segments using EBV-LCL feeder cells in advance of thawing of the cord unit. These methods are being optimized to allow for clinical scale expansion of NK cells from the same cord unit used for hematopoietic cell transplantation. Cell numbers after expansion of individual segments with EBV – LCL feeder cells Day 0 (×10e6) Day 16 (× 10e6) Fold expansion TNC 3–6 78–112 20–30 CD3+ 0.42–0.91 33–47 50–70 CD3+CD56- 0.015–0.032 16–24 700–1000 CD3-CD56+ 0.081–0.175 28–40 200–300 Cell numbers after CD3+ depletion of pooled segments and expansion with EBV – LCL feeder cells Day 0 (×10e6) Day 33 (× 10e6) Fold expansion TNC 4.16 401 96 CD3+ 0 0 - CD3+CD56- 0 0 - CD3-CD56+ 0.0132 391 29621

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Immunophenotypic, cytotoxic, proteomic and genomic characterization of human cord blood vs. peripheral blood CD56Dim NK cells

Innate Immunity ◽

10.1177/1753425919846584 ◽

2019 ◽

Vol 25 (5) ◽

pp. 294-304 ◽

Cited By ~ 1

Author(s):

Evan Shereck ◽

Nancy S Day ◽

Aradhana Awasthi ◽

Janet Ayello ◽

Yaya Chu ◽

...

Keyword(s):

Cord Blood ◽

Nk Cells ◽

Peripheral Blood ◽

Cell Function ◽

Nk Cell ◽

Chronic Gvhd ◽

Cell Subset ◽

Receptor Expression ◽

Unrelated Donor ◽

Human Cord Blood

Unrelated cord blood (CB) is an excellent alternative as an allogeneic donor source for stem cell transplantation. CB transplantation is associated with lower incidence of severe acute graft versus host disease (GVHD) and chronic GVHD but similar rates of malignant relapse compared with other unrelated donor cell transplants. NK cells are critical innate immune components and the comparison of CB vs. peripheral blood (PB) NK cells is relatively unknown. NK cell receptor expression, cell function, and maturation may play a role in the risk of relapse after CB transplant. We investigated CB vs. PB NK cell subset cytotoxicity, function, receptor expression, and genomic and proteomic signatures. The CB CD56dim compared with PB CD56dim demonstrated significantly increased expression of NKG2A and NKG2D, respectively. CB vs. PB CD56dim NK cells had significantly decreased in vitro cytotoxicity against a variety of non-Hodgkin lymphoma targets. Various proteins were significantly under- and over-expressed in CB vs. PB CD56dim NK cells. Microarray analyses and qRT-PCR in CB vs. PB CD56dim demonstrated significantly increased expression of genes in cell regulation and development of apoptosis, respectively. In summary, CB vs. PB CD56dim NK cells appear to be earlier in development, have decreased functional activity, and increased capacity for programmed cell death, suggesting that CB NK cells require functional and maturational stimulation to achieve similar functional levels as PB CD56dim NK cells.

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Patients’ Peripheral Blood as a Possible Source of Donor-Derived NK Cells for Ex Vivo Expansion and Genetic Modification for Post-Transplant Cellular Immunotherapy In Cord Blood Recipients

Blood ◽

10.1182/blood.v116.21.2094.2094 ◽

2010 ◽

Vol 116 (21) ◽

pp. 2094-2094

Author(s):

Chihaya Imai ◽

Sakiko Yoshida ◽

Takayuki Takachi ◽

Masaru Imamura ◽

Ryosuke Hosokai ◽

...

Keyword(s):

Cord Blood ◽

Nk Cells ◽

Genetic Modification ◽

Peripheral Blood ◽

Nk Cell ◽

Ex Vivo ◽

K562 Cells ◽

Cellular Immunotherapy ◽

Healthy Individuals ◽

Expansion Range

Abstract Abstract 2094 Haploidentical natural killer (NK) cells can induce and consolidate remission in patients with high-risk acute myeloid leukemia (AML) (Rubnitz et al. J Clin Onc 24: 371, 2010). Recently, significantly reduced relapse rates were observed in AML patients who received killer immunoglobulin-like receptor ligand-mismatched cord blood, suggesting effective alloreactivity of cord blood-derived NK cells (Willemze et al. Leukemia 23: 492, 2009). Cord blood transplantation (CBT) is an effective alternative source for allogeneic hematopoietic cell transplantation in both children and adults. However, its therapeutic efficacy for malignant diseases is limited by the lack of available donor effector cells, such as cytotoxic T lymphocytes, lymphokine-activated killer cells, NK-like T cells and NK cells, for treatment of hematological relapse and posttransplant lymphoproliferative disorder and/or for scheduled posttransplant cellular immunotherapy against refractory diseases. We previously reported a method that induces NK cells to proliferate and reliably allows their genetic modification in healthy individuals and leukemia patients in remission receiving maintenance chemotherapy (Imai et al. Blood 106: 376, 2005). To explore the possibility of using patients’ peripheral blood as a source for posttransplant NK cell therapy, we used our method to expand donor-derived NK cells from peripheral blood of CBT recipients early after engraftment. We also examined whether NK cells can be rendered cytotoxic against original leukemia blasts by transferring an antigen-specific artificial immunoreceptor gene. This study was approved by an institutional ethical committee. Patients received CBT for consolidation of hematological malignancy (n=7), neuroblastoma (n=1) or resolution of refractory EBV-associated hemophagocytic syndrome (n=1) with myeloablative (n=7) or reduced intensity conditioning (RIC) regimens (n=2). The patients were enrolled in the study after engraftment and peripheral blood was obtained after appropriate written consent was obtained. A chimerism study using short tandem repeat assays showed complete donor chimerism in all patients except one who received RIC-CBT. The peripheral blood was obtained at a median of 92 days post-CBT (range: 46–303 days) and subjected to ex vivo activation and expansion using a previously described protocol with slight modifications. Briefly, peripheral blood was coincubated with modified K562 cells expressing membrane-bound IL-15 and 4-1BB ligand (K562-mb15-41BBL) in the presence of low-dose IL-2 (10 U/mL). Most patients were on maintenance immunosuppressive therapy with calcineurin inhibitors with (n=3) or without (n=6) systemic corticosteroids. After 7 days of culture, a median 11.0-fold expansion (range: 5.3–28.9-fold) was observed in all but one patient who had been administered chemotherapy with Mylotarg for relapsed AML a few days before the blood sampling. The expansion rate in the first week was less efficient in CBT recipients than in healthy individuals (>20-fold), probably because of the immunosuppressants administered. However, an additional 2-week culture in the presence of high-dose IL-2 (1000 U/mL) yielded a median 206-fold expansion (range: 101–1381-fold in 21 days). The expanded NK cells exhibited upregulation of activating receptors including NKG2D, NCRp30 and NCRp44, and vigorous cytotoxicity against K562 cells (86.8–97.7% at an E/T ratio of 1:1). The NK cells were susceptible to retroviral genetic modification with the MSCV-IRES-GFP vector (median GFP-positive cells, 52.7%, n=10). Finally, peripheral NK cells from patients with acute lymphoblastic leukemia were expanded and transduced with the chimeric immunoreceptor gene anti-CD19-BB-ζ. The donor-derived NK cells expressed large amounts of anti-CD19 chimeric receptors on their surface and killed original leukemia blasts that were highly resistant to NK cell lysis (e.g. anti-CD19 vs. non-signaling receptor: 69% vs. 0% at an E/T ratio of 1:1). These results suggest that, in CBT recipients, ex vivo expansion and genetic modification of donor-derived NK cells from the patients’ peripheral blood is feasible. Because peripheral blood can be easily and repeatedly obtained, the method described here will allow multiple scheduled infusions. This preliminary study may lead to a novel strategy for posttransplant donor-NK cell therapy in CBT recipients. Disclosures: No relevant conflicts of interest to declare.

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