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  • 1
    Online Resource
    Online Resource
    Experimental Double Cord Blood Transplantation (DCBT) of An Expanded Unit Together with a Non-Manipulated Unit in NOD/SCID Mice.
    American Society of Hematology ; 2008
    In:  Blood Vol. 112, No. 11 ( 2008-11-16), p. 2313-2313
    Details
    In: Blood, American Society of Hematology, Vol. 112, No. 11 ( 2008-11-16), p. 2313-2313
    Abstract: We found that nicotinamide (NAM), a form of VitB3 and a recognized inhibitor of SIRT1, the human ortholog of the yeast Sir2 class III NAD+-dependent histone deacetylase, inhibits in vitro differentiation and promotes expansion of hematopoietic progenitor cells. Cord blood (CB)-derived CD34+ cells cultured with cytokines (FLT3, TPO, IL-6, SCF; 50ng/ml) and NAM (2.5mM) (Sigma Aldrich, catalog number N5535) display enhanced in vitro migratory activity toward SDF-1 and home to the BM (24hr following infusion in vivo) with higher efficacy than cells cultured with cytokines only. The number of SCID-repopulating cells increased by 9- and 7.6-fold in cultures treated with NAM relative to non-cultured cells and cytokine only cultured cells, respectively. This net increase in repopulation potential was sustained in competitive transplant experiments where cultured cells were infused along with non-cultured competitor cells derived from the same CB unit. Several experimental clinical protocols of CB-derived expanded cells involve co-transplantation of cultured and non-cultured cells in a double CB transplantation (DCBT). We therefore sought to investigate the engraftment potential of NAM-treated cultured cells in a DCBT setting in NOD/SCID mice, using two CBUs marked as “unit-1” and “unit-2”. Two similar experiments were conducted, each experiment had 4 groups of mice (n=10/ experimental group): non-cultured cells cells cultured with NAM transplanted along with the CD34 negative cell fraction from the same unit that was kept frozen till the day of transplantation. groups a + b non-cultured cells from unit-1 transplanted along with non-cultured-cells from unit-2. Mice were transplanted with similar number of nuclear cells from the cultured or non-cultured units in the single or the DCBT groups. In Experiment-1, non-cultured cells were derived from one unit-1 and NAM-treated cultured cells were derived from another unit-2. In Experiment-2, we switched between the units (such an experiment was possible since our CBUs are frozen in several portions). Level of engraftment was evaluated two weeks post transplantation by FACS analysis of human CD45+ cells while the contribution of each unit to engraftment was measured by quantitative PCR for informative short tandem repeat (qSTR) regions that distinguished the units. The results show that in both experiments, the level of engraftment in the DCBT cohort of a cultured unit transplanted along with a non-cultured unit (12.8±1.8 %) was similar to the level of engraftment of the cultured unit when individually transplanted (15.4 % ±3, p & gt;0.05). This level of engraftment was 9.8-fold higher (p & lt;0.05) than the level of engraftment obtained in the DCBT cohort of two non-cultured units (1.3 % ±0.2). Chimerism analysis indicated that in recipients transplanted with two non-cultured units, engraftment was predominantly derived from unit-1, while in the two experiments of DCBT of a cultured unit transplanted along with a non-cultured unit the engraftment was predominantly derived from the cultured unit. In conclusion, the outstanding engraftment advantage of NAM cultured cells may explain their predominance in a DCBT setting.
    Type of Medium: Online Resource
    ISSN: 0006-4971 , 1528-0020
    URL: Article
    DOI: 10.1182/blood.V112.11.2313.2313
    RVK:
    XA 33000
    RVK:
    XA 10000
    Language: English
    Publisher: American Society of Hematology
    Publication Date: 2008
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  • 2
    Online Resource
    Online Resource
    Nicotinamide, a Recognized Inhibitor of SIRT1, Promotes Expansion in Ex Vivo Cultures of Short and Long-Term Repopulating Cells.
    American Society of Hematology ; 2009
    In:  Blood Vol. 114, No. 22 ( 2009-11-20), p. 2431-2431
    Details
    In: Blood, American Society of Hematology, Vol. 114, No. 22 ( 2009-11-20), p. 2431-2431
    Abstract: Abstract 2431 Poster Board II-408 Nicotinamide (NAM), is a form of VitB3 that recognized and inhibits SIRT1, the human ortholog of the yeast Sir2 class III NAD+-dependent histone deacetylase. We have previously demonstrated that NAM inhibits in vitro differentiation and enhances expansion, migration, homing and NOD/SCID engraftment efficacy of cord blood (CB)-derived CD34+ cells cultured with cytokines. In the current study, the in vivo function of ex vivo cultured cells with NAM was tested in a congenic mice model (BALB/C, CD45.1/CD45.2) for BM transplantation. Purified CD117+ BM cells from BALB/C CD45.1 mice were cultured with a combination of 4 cytokines (FLT3, SCF, TPO, IL-6, 50 ng/ml each), with and without 0.5mM NAM for three weeks. Numbers of CFUc, CD117+ and CD117+Lin- cells were significantly (p & lt; 0.05) higher in cultures treated with NAM as compared with cultured treated with cytokines alone. Non-cultured, freshly purified CD117+ cells (1000 and 2500 cells/mice) and their total progeny following expansion with or without NAM were transplanted into ablated (1000 Rad) CD45.2 mice (n = 10/cohort), 24h post irradiation (Fig 1). Three months post transplantation, all the mice in the control group (non-transplanted) died. The percent survival of mice transplanted with cells cultured with cytokines and NAM was remarkably higher over the survival of mice in the cohort transplanted with cells cultured with cytokines alone or non-cultured cells (Fig 1). FACS analysis (CD45.1-donor / CD45.2-host) of peripheral blood from mice transplanted with NAM cultured cells show 80% donor cell chimerism (CD45.1), 3 and 6 months post transplantation. Percent of donor derived Gr-1+ and CD3+ cells were similar in mice transplanted with non-cultured or NAM cultured cells. Percentages of donor cell chimerism (CD45.1) in secondary mice transplanted with total BM cells derived from primary recipients originally transplanted with non-cultured and NAM cultured cells were 47 and 73, respectively, 6 weeks after the secondary transplantation. In a different experiment, to follow time to engraftment during the first month post transplantation, mice transplanted with non-cultured cells or cells cultured with cytokines and NAM (n = 10/cohort) were bled at weekly intervals and peripheral blood samples were counted for WBC and analyzed by the FACS to determine donor cell chimerism and lineage engraftment. The results show accelerated engraftment (Fig 2) and higher levels of donor cell chimerism (Fig 3) in the cohort transplanted with NAM cultured cells relative to the cohort transplanted with non-cultured cells. Number of granulocytes, T, NK and B cells during the first month post transplant were also significantly (p & lt;0.05) higher in mice transplanted with cells cultured with cytokines and NAM relative to their levels in mice transplanted with non-cultured cells. The results obtained in the congenic mice model for BMT suggest that NAM promotes expansion in ex vivo cultures of short and long-term repopulating cells, as demonstrated by accelerated donor derived engraftment during the first month post transplantation, higher survival of mice, sustained donor cell chimerism 6 month post transplantation and successful reconstitution of secondary recipients. NAM is thus a novel molecule that may be used to stimulate and expand hematopoietic repopulating cells, fasten post transplant engraftment and hopefully improve transplantation outcome. Current studies are designed to elucidate NAM mode of action. Fig 1: Three month survival Fig 1:. Three month survival Fig 2: Short-term Engrafoment Fig: 3 Percentage of Donor Chimerism Fig 2:. Short-term Engrafoment Fig: 3 Percentage of Donor Chimerism Disclosures: Peled: Gamida-Cell: Employment, Equity Ownership. Shoham:Gamida Cell: Employment. Aschengrau:Gamida Cell: Employment. Yackoubov:Gamida Cell: Employment. Frei:Gamida Cell: Employment. Nagler:Gamida Cell: Arnon Nagler, Consultancy. Peled:Gamida Cell: Consultancy.
    Type of Medium: Online Resource
    ISSN: 0006-4971 , 1528-0020
    URL: Article
    DOI: 10.1182/blood.V114.22.2431.2431
    RVK:
    XA 33000
    RVK:
    XA 10000
    Language: English
    Publisher: American Society of Hematology
    Publication Date: 2009
    detail.hit.zdb_id: 1468538-3
    detail.hit.zdb_id: 80069-7
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  • 3
    Online Resource
    Online Resource
    Nicotinamide, a SIRT1 inhibitor, inhibits differentiation and facilitates expansion of hematopoietic progenitor cells with enhanced bone marrow homing and engraftment
    Elsevier BV ; 2012
    In:  Experimental Hematology Vol. 40, No. 4 ( 2012-04), p. 342-355.e1
    Details
    In: Experimental Hematology, Elsevier BV, Vol. 40, No. 4 ( 2012-04), p. 342-355.e1
    Type of Medium: Online Resource
    ISSN: 0301-472X
    URL: Article
    DOI: 10.1016/j.exphem.2011.12.005
    RVK:
    XA 42470
    Language: English
    Publisher: Elsevier BV
    Publication Date: 2012
    detail.hit.zdb_id: 2005403-8
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  • 4
    Online Resource
    Online Resource
    Nicotinamide Modulates Ex-Vivo Expansion of Cord Blood Derived CD34+ Cells Cultured with Cytokines and Promotes Their Homing and Engraftment in SCID Mice.
    American Society of Hematology ; 2006
    In:  Blood Vol. 108, No. 11 ( 2006-11-16), p. 725-725
    Details
    In: Blood, American Society of Hematology, Vol. 108, No. 11 ( 2006-11-16), p. 725-725
    Abstract: Nicotinamide (NA) is a non-competitive inhibitor of NAD(+)-dependent ADP-ribosyl transferases, of CD38 NADase (a major regulator of cellular NAD levels) and of Sir2 histone-deacetylase. These enzymes are playing a pivotal role in regulation of signal transduction pathways and gene expression. In the present study, we evaluated the effect of NA on the ex-vivo expansion of cord-blood (CB) derived CD34+ cells and their bone-marrow (BM) homing and engraftment potential. Culturing of CD34+ cells for three weeks in the presence of cytokines (SCF, TPO, IL-6, FLT3-ligand) only or cytokines + NA (5mM) resulted in similar expansion of CD34+ cells (40-fold relative to input). However, a remarkable increase in the fraction of CD34+ cells displaying an early progenitor cell phenotype (CD34+Lin−) was observed in the NA-treated cultures as compared with cytokines-only treated cultures (18.6 ± 3% and 0.7 ± 0.06%, n=6, p & lt;0.05, respectively). Tracking the cell-cycle history by PKH2 staining showed fewer division cycles of CD34+ cells cultured with NA. These results may suggest a direct correlation between the rate of proliferation and expansion of CD34+Lin− cells. NA-treated CD34+ cells express similar levels of CXCR4 but display increased migratory activity in response to CXCL12 over CD34+ cells treated with cytokines only (36 ± 19% and 11 ± 4%, n=4, p & lt;0.05, respectively). In order to test their homing potential, similar number of mononuclear cells (MNC), before or following expansion with or without NA, were labeled with CFSE and transplanted into irradiated NOD/SCID mice. Twenty-four hours later the numbers of human cells (CD45+CFSE+) and human progenitor cells (CD34+CFSE+) in the BM were counted. Homing of CD45+CFSE+ cells was comparable in the three groups tested. However, CD34+CFSE+ cells with BM homing potential were 3-fold more numerous in NA-treated cultures relative to cytokines-treated cultures, and 6-fold more than in non-cultured CB cells (n=14, p & lt;0.05). To evaluate engraftment, SCID mice were transplanted with 3x103, 6x103 and 12x103 non-cultured CD34+ cells or their entire progeny following 3-week expansion with cytokines only or cytokines + NA (n = 63). The frequency of SCID repopulating cells (SRC) was estimated by limiting dilution analysis as 1/ 36,756 (non-cultured), 1/19,982 (cytokines), 1/ 2,620 (NA) (SCID engraftment was considered as ≥0.5% human CD45+ cells). We found that, in correlation with homing, NA-treated cells have a 14- and 7.6-fold more SRC than non-cultured cells or cytokine-treated cells, respectively. The marked increase in SCID engraftment potential following culturing with NA may be attributed to both improved homing of CD34+ cells as well as higher proportion of early progenitor cells within the CD34+ cell compartment. Despite their numerical expansion, progenitor cells generated in cytokine-supplemented cultures have reduced homing and engraftment capacity. Our study demonstrates that NA modulates in-vitro expansion and augments the in-vivo homing and engraftment of CB-derived CD34+ cells cultured with cytokines.
    Type of Medium: Online Resource
    ISSN: 0006-4971 , 1528-0020
    URL: Article
    DOI: 10.1182/blood.V108.11.725.725
    RVK:
    XA 33000
    RVK:
    XA 10000
    Language: English
    Publisher: American Society of Hematology
    Publication Date: 2006
    detail.hit.zdb_id: 1468538-3
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  • 5
    Online Resource
    Online Resource
    Nicotinamide (NAM) Modulates Transcriptional Signature of Ex Vivo Cultured UCB CD34+ Cells (Omidubicel) and Preserves Their Stemness and Engraftment Potential
    American Society of Hematology ; 2019
    In:  Blood Vol. 134, No. Supplement_1 ( 2019-11-13), p. 3718-3718
    Details
    In: Blood, American Society of Hematology, Vol. 134, No. Supplement_1 ( 2019-11-13), p. 3718-3718
    Abstract: Historical efforts at expansion of umbilical cord blood (UCB) derived CD34+ hematopoietic stem cells (HSCs) ex vivo with cytokines yielded large numbers of progenitors for transplantation but impaired their long-term engraftment ability. We used nicotinamide (NAM), an allosteric inhibitor of NAD-enzymes, to create omidubicel, an investigational cell therapy designed to improve the expansion of CD34+ HSCs for bone marrow transplant. A Phase 1/2 clinical study of omidubicel in patients with high-risk hematologic malignancies showed rapid neutrophil engraftment and a more favorable immune reconstitution profile in patients compared to historical controls.1 We hypothesized that NAM treatment maintains the stemness and engraftment potential of omidubicel, which is associated with clinical benefit.2 We performed transcriptome, transcription factor (TF), and pathway analysis by next generation sequencing (NGS) to discern the mechanism of action of NAM and to elucidate the pathways leading to the preservation of engraftment after ex vivo expansion of omidubicel compared to CD34+ cells grown in the absence of NAM. Transcriptome analysis revealed that treatment of CD34+ cells with cytokines alone (stem cell factor [SCF], thrombopoietin [TPO] , IL-6, and FLT3 ligand) led to an increase in pathways responsible for cell proliferation and differentiation, apoptotic stress, and production of reactive oxygen species (ROS), and matrix metalloproteinases (MMPs), all of which were attenuated by NAM. TF enrichment analysis of NAM-upregulated genes and downregulated genes demonstrated that NAM modulated several TFs critically involved in pathways of HSC cell self-renewal, differentiation, apoptosis and migration. Specifically, NF-kB, C-Jun, LXR/RXR and PPARα/RXRα, and AMPK-mTor signaling were all reduced in NAM-treated CD34+ cells compared to controls. Reduced expression of key genes involved in the production of ROS and reactive nitrogen species (RNS) including NADPH-oxidase-related genes (CYBB, NCF2 and NCF4) and iNOS, suggested that NAM-expanded CD34+ cells were less exposed to oxygen and nitrogen free radical stress than controls. NAM also downregulated the expression of several matrix metalloproteinases (MMP) genes including MMP7, MMP9, MMP12 and MMP19. NAM-induced downregulation of MMPs may explain the increase in engraftment in patients receiving omidubicel. Pathway analysis of differentially expressed (DE) genes was conducted using ingenuity (IPA) software. IPA analysis of DE genes showed significant downregulation of growth factor activating pathways including SCF, TPO, FLT, and GM-CSF and Endothelin-1 and P2Y Purigenic Receptor, which was confirmed by a reduction in cell cycling rates of labeled cells. IPA analysis also pointed to genes in 3 key cellular pathways that were downregulated by NAM: stress induction of apoptosis, production of ROS and RNS, and production of MMPs. NAM treatment also uniquely upregulated genes linked to cellular metabolism including the Sirtuin family genes, TCA cycle genes, and HIF1a. Interestingly, NAM upregulated genes responsible for telomerase expression further validating our hypothesis that NAM preserves cell stemness. In summary, NGS transcriptome analysis revealed that ex vivo expansion of UCB derived CD34+ cells in the presence of NAM attenuated TFs responsible for proliferation and differentiation of stem cells. In addition, NAM treatment downregulated genes regulating the production ROS, RNS, and MMPs and upregulated genes controlling metabolism and senescence, thus allowing for the expansion of CD34+ cells with preserved function and long-term engraftment ability. Our gene expression data leads to a better understanding of the mechanisms by which NAM modulates CD34+ cells in omidubicel to preserve their function. These data provide further scientific rationale for the favorable clinical engraftment and patient outcomes observed in the Phase 1/2 clinical study of omidubicel.1 An international, randomized, multi-center Phase 3 study of omidubicel in patients with high-risk hematologic malignancies is underway.2 [1]Horwitz M.E., et. al., J Clin Oncol. 2019 Feb 10;37(5):367-374. [2] ClinicalTrials.gov identifier NCT02730299. Disclosures Lodie: Gamida Cell: Employment, Equity Ownership. Adams:Gamida Cell: Employment, Equity Ownership. Yackoubov:GAMIDA CELL: Employment, Other: unexecuted shares of the company . Peled:Gamida Cell: Employment, Equity Ownership.
    Type of Medium: Online Resource
    ISSN: 0006-4971 , 1528-0020
    URL: Article
    DOI: 10.1182/blood-2019-131124
    RVK:
    XA 33000
    RVK:
    XA 10000
    Language: English
    Publisher: American Society of Hematology
    Publication Date: 2019
    detail.hit.zdb_id: 1468538-3
    detail.hit.zdb_id: 80069-7
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  • 6
    Online Resource
    Online Resource
    Nicotinamide, a Form of Vitamin B3, Promotes Expansion of Natural Killer Cells That Display Increased In Vivo Survival and Cytotoxic Activity,
    American Society of Hematology ; 2011
    In:  Blood Vol. 118, No. 21 ( 2011-11-18), p. 4035-4035
    Details
    In: Blood, American Society of Hematology, Vol. 118, No. 21 ( 2011-11-18), p. 4035-4035
    Abstract: Abstract 4035 NK cells are cytotoxic lymphocytes that have drawn considerable attention in recent years as a promising tool for immunotherapy in patients with various refractory hematological malignancies and metastatic solid tumors. Clinical results of experimental protocols have shown only a partial response attributed mainly to the relatively low number of NK cells infused and their short in vivo persistence. An important challenge, therefore, in advancing the clinical applicability of NK cells is to expand ex vivo NK cells that display increased functionality upon in vivo infusion. In efforts to induce NK cell expansion, different combinations of cytokines have been studied. However, most reports show a modest expansion and demonstrate a need for additional stimuli. Nicotinamide (NAM) is a form of Vitamin B3 and a potent inhibitor of enzymes that use NAD for their activity. Hence, NAM is directly involved in the control of redox sensitive enzymes, mitochondrial functions, cell metabolism, the production of energy, and cell motility. Here we show that NAM (2.5–5 mM) enhances expansion (60-80 fold) of functional NK cells in feeder-free cultures stimulated with IL-2 and IL-15 for two weeks. This effect was observed in cultures initiated with purified CD56+ (CD56 enriched/CD3 depleted) or with CD3 depleted, peripheral blood and cord blood cells. Immunophenotyping of the cultured NK cells has so far revealed that NAM substantially modulates three cell surface receptors. CD200R and programmed death receptor-1 (PD-1) expressed on NK cells interact with their ligands on tumor cells which leads to a suppression in NK cell anti-tumor activity and tumor immunoevasion. These two receptors are down-regulated by NAM. CD62L (L-selectin) defines an NK subset with increased self-renewal capacity and its expression was reported to be pivotal for NK cells trafficking to lymphoid organs and their homeostatic proliferation. Following expansion in culture with IL-2, CD62L is down-regulated, whilst NAM increased its expression with a dose-dependent effect. Using a CFSE-based cytotoxicity assay we have demonstrated that NK cells cultured with NAM display higher cytotoxic activity against K562, BL2, NK-resistant COLO 205 cell lines and primary leukemia cells. In a Transwell migration assay, NK cells cultured with NAM demonstrated increased migration towards the CXCR4 ligand SDF-1. To test in vivo homing and retention, irradiated (350 RAD) NOD/SCID mice were transplanted with a similar number of cells (15–20×106 /mouse) derived from two week cultures treated with or without NAM. Mice were infused with 50μg/mouse IL-2 and 5μg/mouse IL-15 every other day. To test homing, mice were sacrificed 24 hour post infusion. Number of human NK cells (CD45+CD56+) detected in the spleen and BM were significantly (p 〈 0.05) higher in the cohort of mice infused with NK cells cultured with NAM (7.9 and 1.39 respectively) compared to mice infused with NK cells cultured without NAM (4.13 and 0, respectively). In a different set of experiments, persistence of human NK cells was analyzed 4 and 12 days post infusion. Four days post infusion, the percentage of human NK cells in the spleen, BM, lung and liver were substantially higher in mice infused with NK cells cultured with NAM compared to mice infused with NK cells cultured without NAM (Fig 1). Even though 12 days post infusion, a decrease in the number of human NK cells was observed in comparison to day 4, still cell retention in the spleen, liver and lung was significantly greater in the cohort infused with NK cells cultured with NAM (13.45, 0.6, 9.21% Vs. 1.26, 0.12, 2.85%, (p 〈 0.05), respectively). The calculated decrease in the number of human NK cells from day 4 to 12 was 50% less in the NAM cohort, suggesting enhanced in vivo survival of NK cells cultured with NAM.Table 1:In vivo persistence of ex vivo expanded NK cellsTable 1:. In vivo persistence of ex vivo expanded NK cells In conclusion, expansion of NK cells with NAM was found to increase in vivo homing and survival and to augment tumor cytotoxic effect of NK cells. This suggests a potential for enhancing the clinical efficacy of adoptively transferred NK cells. Based on these intriguing findings we are developing a cell product for adoptive cell-mediated immune therapy. Disclosures: Frei: Gamida Cell: Employment. Persi:Gamida Cell: Employment. Lador:Gamida Cell: Employment, Equity Ownership. Peled:Gamida Cell: Consultancy. Nagler:Gamida Cell: Consultancy. Peled:Gamida Cell: Employment, Equity Ownership, Patents & Royalties.
    Type of Medium: Online Resource
    ISSN: 0006-4971 , 1528-0020
    URL: Article
    DOI: 10.1182/blood.V118.21.4035.4035
    RVK:
    XA 33000
    RVK:
    XA 10000
    Language: English
    Publisher: American Society of Hematology
    Publication Date: 2011
    detail.hit.zdb_id: 1468538-3
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  • 7
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    Online Resource
    Improved Homing To Bone Marrow, Spleen and Lung Of Adoptively Infused NK Cells Expanded Ex Vivo With The Small Molecule Nicotinamide Using Feeder-Free Conditions
    American Society of Hematology ; 2013
    In:  Blood Vol. 122, No. 21 ( 2013-11-15), p. 897-897
    Details
    In: Blood, American Society of Hematology, Vol. 122, No. 21 ( 2013-11-15), p. 897-897
    Abstract: Clinical results with NK cells in investigational tumor immunotherapy protocols have at best resulted in partial responses only. The inability of ex vivo expanded NK cells to proliferate in vivo, as well as to home to and be retained in the tumor micro-environment, likely plays a role in their limited efficacy to date. Clinical grade NK cells expanded using EBV-LCL feeder cells (FC) have recently been evaluated in the clinic (NHLBI) for hematological malignancies and metastatic cancers with only minor responses being observed to date. We observed that CD62L expression was down-regulated on EBV-LCL NK cells compared to fresh NK cells, potentially limiting their clinical activity. Since NAM up-regulates CD62L on NK cells cultured in feeder-free (FF) conditions, we compared the ability of FF NK cells with EBV-LCL NK cells to home and persist in vivo following adoptive transfer into NSG mice. FF NK cultures were initiated with CD3 depleted PB TNC while EBV-LCL NK cell cultures were initiated by co-culturing 100 cGy-irradiated SMI-EBV-LCL FC with CD3 depleted, CD56 enriched cells. NSG mice received 200 cGy of TBI followed 24 hours later by 10 million IV NK cells. Three cohorts were studied: a) NK cells expanded using EBV-LCL FC; b) FF expanded NK cells without NAM and c) FF expanded NK cells with NAM (5mM). All NK cell cohorts were expanded from the same human donor. Cells were harvested from the blood, lungs, spleen, and BM of mice 4 days after infusion (n=5). NK cells expanded with NAM in FF conditions were detectable in all mouse organs including the PB at significantly higher levels than NK cells expanded in FF conditions without NAM or using EBV-LCL FC (Fig.1a). Subsequent experiments transferring CFSE-labeled NK cells expanded with NAM into irradiated NSG mice showed a marked reduction in CFSE intensity and the presence of multiple peaks after 4 days, indicative of in vivo proliferation. We next evaluated the impact of daily exogenous IL-2 or IL-15 administration on the homing potential of expanded NK cells 4 days post-infusion into irradiated NSG mice (n=5). We observed that both IL-2 and IL-15 enhanced homing of NK cells expanded with NAM in FF conditions, but failed to enhance the homing of NK cells expanded with EBV-LCL FC (Fig. 1b). With the exception of CD62L, no consistent differences in the phenotype of NK cells between the various expansion methods were observed. NK cells expanded in all groups maintained similar levels of cytotoxicity against multiple different targets including K562 cells, myeloma and renal cell carcinoma tumor cell lines. The number of NK cells expanded using FF conditions was lower than NK cells expanded with EBV-LCL. Nevertheless, NK cells cultured in NAM without feeder cells still expanded a median 50 (37-87) fold, yielding a total of 140x108 NK cells (purity 〉 98%) from a single aphaeresis collection. Further, cytokine levels measured from the supernatants of NK cells cultured with tumor targets showed significantly higher levels of IFNγ, TNFα and FAS-L secretion from FF NK cells expanded with NAM in comparison to the other two groups (Fig1c). Conclusion These data show human NK cells expanded ex vivo in NAM utilizing FF conditions substantially up-regulate CD62L, have enhanced inflammatory cytokine secretion against tumors, and have improved in vivo proliferation and homing to multiple organs including the bone marrow compared to EBV-LCL expanded NK cells. These differences suggest NAM expanded NK cells could have superior clinical efficacy compared to EBV-LCL expanded NK cells following adoptive transfer into patients with hematological malignancies and metastatic cancers. Frei: Gamida Cell: Employment. Peled:Gamida Cell: Employment. Persi:Gamida Cell: Employment. Lador:Gamida Cell: Employment. Peled:Gamida Cell: Consultancy. Nicotinamide (NAM) is a small molecule form of Vitamin B3 and a potent inhibitor of enzymes that use NAD for their activity and thus is involved in the control of redox-sensitive enzymes, mitochondrial functions, cell metabolism and production of energy and cell motility. NAM, when used as an epigenetic modulator has been shown to increase the homing and engraftment efficacy to the BM of ex vivo expanded CD34+ cells. Recently, we found (Gamida-Cell) that NAM also enhances the in-vivo homing and retention of peripheral blood (PB) derived NK cells expanded over two weeks in feeder-free culture conditions stimulated with IL-2 or IL-15. Immunophenotype studies demonstrated NAM-treated cultures had a substantial increase in CD62L (L-selectin), pivotal for NK cell trafficking and homeostatic proliferation.
    Type of Medium: Online Resource
    ISSN: 0006-4971 , 1528-0020
    URL: Article
    DOI: 10.1182/blood.V122.21.897.897
    RVK:
    XA 33000
    RVK:
    XA 10000
    Language: English
    Publisher: American Society of Hematology
    Publication Date: 2013
    detail.hit.zdb_id: 1468538-3
    detail.hit.zdb_id: 80069-7
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  • 8
    Online Resource
    Online Resource
    Nicotinamide (NAM) Modulates Transcriptional Signature of Ex Vivo Cultured UCB CD34+ Cells (Omidubicel) and Preserves Their Stemness and Engraftment Potential
    Elsevier BV ; 2020
    In:  Biology of Blood and Marrow Transplantation Vol. 26, No. 3 ( 2020-03), p. S258-S259
    Details
    In: Biology of Blood and Marrow Transplantation, Elsevier BV, Vol. 26, No. 3 ( 2020-03), p. S258-S259
    Type of Medium: Online Resource
    ISSN: 1083-8791
    URL: Article
    DOI: 10.1016/j.bbmt.2019.12.453
    Language: English
    Publisher: Elsevier BV
    Publication Date: 2020
    detail.hit.zdb_id: 3056525-X
    detail.hit.zdb_id: 2057605-5
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  • 9
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    Enhanced In Vivo Persistence and Proliferation of NK Cells Expanded in Culture with the Small Molecule Nicotinamide: Development of a Clinical-Applicable Method for NK Expansion
    American Society of Hematology ; 2017
    In:  Blood Vol. 130, No. Suppl_1 ( 2017-12-07), p. 657-657
    Details
    In: Blood, American Society of Hematology, Vol. 130, No. Suppl_1 ( 2017-12-07), p. 657-657
    Abstract: Adoptive transfer of cytolitic Natural Killer (NK) cells is a promising immunotherapeutic modality for hematologic and other malignancies. However, limited NK cell in vivo persistence and proliferation have been challenging clinical success of this therapeutic modality. Here we present a reliable, scalable and GMP-compliant culture method for the expansion of highly functional donor NK cells for clinical use. Nicotinamide (NAM), a form of vitamin B-3, serves as a precursor of nicotinamide adenine dinucleotide (NAD) and is a potent inhibitor of enzymes that require NAD including ADP ribosyltransferases and cyclic ADP ribose/NADase. As such, NAM is implicated in the regulation of cell adhesion, polarity, migration, proliferation, and differentiation. We have previously reported that NAM augments tumor cytotoxicity and cytokine (TNFα and IFN-γ) secretion of NK cells expanded in feeder-free culture conditions stimulated with IL-2 or IL-15. Immunophenotype studies demonstrated NK cells expanded with NAM underwent typical changes observed with cytokine only-induced NK cell activation with no significant differences in the expression of activating and inhibitory receptors. CD200R and PD-1 receptors were expressed at low levels in resting NK cells, but their expression was up-regulated following activation in typical cytokine expansion cultures. Interestingly, the increase in CD200R and PD-1 was reduced by NAM, suggesting these NK cells to be less susceptible to cancer immunoevasion mechanisms (Fig 1). In vivo retention and proliferation is a pre-requisite for the success of NK therapy. We have reported that NK expanded with NAM displayed substantially better retention in the bone marrow, spleen and peripheral blood of irradiated NSG mice. Using a carboxyfluorescein succinimidyl ester (CFSE) dilution assay, we demonstrated increased in vivo proliferation of NAM-cultured NK cells compared with cells cultured without NAM. These results were recently confirmed using a BrdU incorporation assay in irradiated NSG mice (Fig.2). These findings were mechanistically supported by a substantial increase in CD62L (L-selectin) expression in cultures treated with NAM. CD62L is pivotal for NK cell trafficking and homeostatic proliferation and its expression is down regulated in IL-2 or IL-15 stimulated cultures (Fig. 3). These data provided the foundation for the development of a feeder cell-free scalable culture method for clinical therapy using apheresis units obtained from healthy volunteers. CD3+ cells were depleted using a CliniMACS T cell depletion set. Following depletion, the CD3- fraction was analyzed for phenotypic markers and cultured in closed-system flasks (G-Rex100 MCS, Wilson Wolf) supplemented with 20ng/ml IL-15 or 50ng/ml IL-2 GMP, 10% human serum, minimum essential medium-α and NAM USP for two weeks. While at seeding, NK cells comprised 5-20% of total culture seeded cells, at harvest, NK cells comprised more than 97% of the culture. Although overall contamination of the NK cultures was low with either IL-15 or IL-2, a lower fraction of CD3+ and CD19+ cells was observed with IL-15 vs IL-2 (0.2±0.1% vs. 0.4±0.2% and 1.3±0.4% vs. 2.4±0.6%, respectively). Consequently, we decided to use IL-15 for clinical manufacturing. Optimization of NAM concentration studies showed similar expansion with 2.5 and 5 mM and a decrease in expansion with 7.5 mM NAM. Since NAM at 5 mM had a stronger impact on CD62L expression and on the release of IFNγ and TNFα than NAM at 2.5 mM, we selected 5mM NAM for clinical manufacturing. Overall median NK expansion after two weeks in closed G-Rex flasks supplemented with IL-15 and 5mM NAM was 50-fold (range 37-87). An additional and significant increase in expansion was obtained after doubling the culture medium one week post seeding. While there was a marked advantage for single culture feeding, more feedings had less impact on NK expansion and had a negative effect on the in vivo retention potential. Our optimized expansion protocol therefore involved one feeding during the two weeks expansion duration resulting in 162±30.7-fold expansion of NK cells relative to their input number in culture. Based on these data, we have initiated a clinical trial at University of Minnesota, to test the safety and efficacy of escalating doses (2 x 107/kg - 2 x 108/kg) of our novel NAM NK cell product in patients with refractory non-Hodgkins lymphoma and multiple myeloma (NCT03019666). Disclosures Peled: Gamida Cell: Employment, Equity Ownership. Brachya: Gamida Cell: Employment. Persi: Gamida Cell: Employment. Lador: gamida Cell: Employment, Equity Ownership. Olesinski: gamida cell: Employment. Landau: gamida cell: Employment, Equity Ownership. Galamidi: gamida cell: Employment. Peled: Biokine: Consultancy; Biosight: Consultancy. Miller: Celegene: Consultancy; Oxis Biotech: Consultancy; Fate Therapeutics: Consultancy, Research Funding. Bachanova: Oxis: Membership on an entity's Board of Directors or advisory committees, Research Funding; Zymogen: Consultancy, Membership on an entity's Board of Directors or advisory committees; Seattle-Genetics: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novartis Pharmaceuticals Corporation: Membership on an entity's Board of Directors or advisory committees, Research Funding; Juno: Membership on an entity's Board of Directors or advisory committees.
    Type of Medium: Online Resource
    ISSN: 0006-4971 , 1528-0020
    URL: Article
    DOI: 10.1182/blood.V130.Suppl_1.657.657
    RVK:
    XA 33000
    RVK:
    XA 10000
    Language: English
    Publisher: American Society of Hematology
    Publication Date: 2017
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    detail.hit.zdb_id: 80069-7
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    Umbilical cord blood expansion with nicotinamide provides long-term multilineage engraftment
    American Society for Clinical Investigation ; 2014
    In:  Journal of Clinical Investigation Vol. 124, No. 7 ( 2014-7-1), p. 3121-3128
    Details
    In: Journal of Clinical Investigation, American Society for Clinical Investigation, Vol. 124, No. 7 ( 2014-7-1), p. 3121-3128
    Type of Medium: Online Resource
    ISSN: 0021-9738
    URL: Article
    URL: Article
    DOI: 10.1172/JCI74556
    DOI: 10.1172/JCI74556DS1
    Language: English
    Publisher: American Society for Clinical Investigation
    Publication Date: 2014
    detail.hit.zdb_id: 2018375-6
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