scholarly journals Preclinical Development of Inducible MyD88/CD40 (iMC)-Enhanced Chimeric Antigen Receptor Natural Killer (GoCAR-NK) Cells to Target BCMA+ Tumors

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 39-40
Author(s):  
Xiaomei Wang ◽  
MyLinh T Duong ◽  
Alan D. Guerrero ◽  
Aruna Mahendravada ◽  
Kelly L Sharp ◽  
...  
Keyword(s):  
Nk Cells ◽  
Cytokine Production ◽  
Nk Cell ◽  
Target Cells ◽  
Publicly Traded ◽  
Nsg Mice ◽  
Co Activation ◽  
Tumor Cytotoxicity

Background: Natural Killer (NK) cells possess potent innate anti-tumor cytotoxicity that can be augmented and focused by engineering with chimeric antigen receptors (CARs). Because NK cells do not express T cell receptors that can direct alloreactivity, they have potential as an off-the-shelf (OTS) cell therapy for the treatment of cancer. We recently demonstrated that a drug-inducible co-activation molecule (inducible MyD88/CD40; iMC) synergizes with transgenic IL-15 to boost CAR-NK cell proliferation, survival and anti-tumor cytotoxic effects (Blood Adv.4:1950 [2020]). Here, we describe the pre-clinical development of an OTS iMC/IL-15-enhanced CAR-NK cell platform targeting B cell maturation antigen (BCMA) for the treatment of multiple myeloma. Methods: NK cells were isolated from peripheral blood mononuclear cells by CD56+ selection, activated with IL-15 and microparticles conjugated with IL-21 and 4-1BB ligand. Activated NK cells were transduced with retrovirus encoding an optimized iMC and IL-15-expressing BCMA CAR construct (iMC-BCMA.z-IL15) where iMC signaling could be activated by exposure to rimiducid (Rim), a small molecule dimerizing ligand. Anti-tumor cytotoxicity and cytokine production was assessed using co-culture assays with control or modified CAR-NK cells against BCMA-expressing myeloma cells (NCIH929, RPMI8226, MM1S, U266 and NALM-6-BCMA). Additional experiments were performed with BCMA-edited cell lines (CRISPR/Cas9) to evaluate the innate cytotoxic potential of GoCAR-NK cells. In vivo anti-tumor efficacy and NK cell expansion was measured using immunodeficient NSG mice engrafted with 1.5 x 106 NCIH929-GFPffluc, MM1S-GFPffluc or THP1-GFPffluc cells followed by i.v. treatment with up to 1 x 107 BCMA GoCAR-NK cells. Tumor and NK cells were tracked via bioluminescence imaging. Results: Following IL-15 and IL-21/4-1BBL microparticle stimulation, NK cells were efficiently transduced (40-70%) and exhibited rapid ex vivo expansion (200-fold in 13 days). iMC-BCMA.CAR-IL15-modified NK cells exhibited potent cytotoxicity against BCMA+target cells compared with mock-transduced NK cells (MM1S, 58±4% versus 17±2%; Nalm-6-BCMA, 61±2% versus 19±6%) after 24 hours. Long-term (7 day) co-culture assays revealed the effect of iMC/IL-15 enhancement on NK cell potency, proliferation and cytokine production where iMC-BCMA.z-IL15-modified NK cells stimulated with Rim showed a >70% increase in tumor-specific killing compared to cells without iMC activation. Further, rimiducid-induced activation led to NK cell persistence and proliferation, 8.1±4.0-fold expansion compared to the start of the coculture. In comparison, there was an 80% reduction mock transduced NK cells or GoCAR-NK cells in cocultures without rimiducid. Induced-MC signaling also drove production of cytokines such as TNF-α, IFN-g (6.6X stimulation with 1 nM Rim relative to no drug), GMCSF, IP-10, and IL-13. In addition, activation of the iMC co-activation protein in combination with IL-15 secretion prevented NK cell exhaustion and led to retained functional activity of the modified GoCAR-NK cells for over 4-weeks in culture. In contrast, unmodified NK cells or modified GoCAR-NK cells without Rim exposure became functionally deficient. Of interest, a comparison of NK and T cells modified with the iMC/IL-15 BCMA CAR construct indicated that CAR-NK cells display more rapid target killing, which is further augmented by iMC-mediated cell signaling in the presence of Rim. Furthermore, GoCAR-NK cells were capable of lysis of BCMA-null target cells due to their innate anti-tumor activity. In vivo efficacy studies showed that neither iMC activation nor IL-15 secretion alone were sufficient to support CAR-NK cell engraftment in NSG mice but, in combination, they resulted in CAR-NK cell expansion and persistence. iMC/IL-15-enhanced BCMA GoCAR-NK cells proliferation was associated with improved control of tumor outgrowth in mice challenged with BCMA+ myeloma cells. Summary: These results indicate that the synergistic activity of iMC signaling combined with transgenic IL-15 production can enhance BCMA-specific CAR-NK cytotoxicity, cytokine production, long-term proliferation and persistence and may improve overall anti-tumor efficacy of a potential OTS cell therapy for the treatment of myeloma. Disclosures Wang: Bellicum Pharmaceuticals: Current Employment, Current equity holder in publicly-traded company. Duong:Bellicum Pharmaceuticals: Current Employment, Current equity holder in publicly-traded company. Guerrero:Bellicum Pharmaceuticals: Current Employment, Current equity holder in publicly-traded company. Mahendravada:Bellicum Pharmaceuticals: Current Employment, Current equity holder in publicly-traded company. Sharp:Bellicum Pharmaceuticals: Current Employment, Current equity holder in publicly-traded company. Brandt:Bellicum Pharmaceuticals: Current Employment, Current equity holder in publicly-traded company. Gagliardi:Bellicum Pharmaceuticals: Current Employment, Current equity holder in publicly-traded company. Foster:Bellicum Pharmaceuticals: Current Employment, Current equity holder in publicly-traded company. Bayle:Bellicum Pharmaceuticals: Current Employment, Current equity holder in publicly-traded company.

scholarly journals CD33 Delineates Two Functionally Distinct NK Cell Populations Divergent in Cytokine Production and Antibody-Mediated Cellular Cytotoxicity

2022 ◽  
Vol 12 ◽  
Author(s):  
Maryam Hejazi ◽  
Congcong Zhang ◽  
Sabrina B. Bennstein ◽  
Vera Balz ◽  
Sarah B. Reusing ◽  
...  
Keyword(s):  
Nk Cells ◽  
Cytokine Production ◽  
Nk Cell ◽  
Functional Divergence ◽  
Target Cells ◽  
Cellular Cytotoxicity ◽  
Transcriptional Signature ◽  
Promising Target

The generation and expansion of functionally competent NK cells in vitro is of great interest for their application in immunotherapy of cancer. Since CD33 constitutes a promising target for immunotherapy of myeloid malignancies, NK cells expressing a CD33-specific chimeric antigen receptor (CAR) were generated. Unexpectedly, we noted that CD33-CAR NK cells could not be efficiently expanded in vitro due to a fratricide-like process in which CD33-CAR NK cells killed other CD33-CAR NK cells that had upregulated CD33 in culture. This upregulation was dependent on the stimulation protocol and encompassed up to 50% of NK cells including CD56dim NK cells that do generally not express CD33 in vivo. RNAseq analysis revealed that upregulation of CD33+ NK cells was accompanied by a unique transcriptional signature combining features of canonical CD56bright (CD117high, CD16low) and CD56dim NK cells (high expression of granzyme B and perforin). CD33+ NK cells exhibited significantly higher mobilization of cytotoxic granula and comparable levels of cytotoxicity against different leukemic target cells compared to the CD33− subset. Moreover, CD33+ NK cells showed superior production of IFNγ and TNFα, whereas CD33− NK cells exerted increased antibody-dependent cellular cytotoxicity (ADCC). In summary, the study delineates a novel functional divergence between NK cell subsets upon in vitro stimulation that is marked by CD33 expression. By choosing suitable stimulation protocols, it is possible to preferentially generate CD33+ NK cells combining efficient target cell killing and cytokine production, or alternatively CD33− NK cells, which produce less cytokines but are more efficient in antibody-dependent applications.

scholarly journals Human CD56bright NK Cells Acquire Potent Anti-Leukemia Functionality Following IL-15 Priming

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 550-550
Author(s):  
Julia A Wagner ◽  
Rizwan Romee ◽  
Maximillian Rosario ◽  
Melissa M Berrien-Elliott ◽  
Stephanie E Schneider ◽  
...  
Keyword(s):  
Nk Cells ◽  
Cancer Immunotherapy ◽  
Target Cell ◽  
Cytokine Production ◽  
Nk Cell ◽  
Cell Subset ◽  
Fold Increase ◽  
Target Cells ◽  
Ifn Γ

Abstract Natural killer (NK) cells are innate lymphoid cells that mediate anti-tumor responses via cytotoxicity and effector cytokine production. Human NK cells are divided into two subsets based on relative expression of CD56 (CD56bright and CD56dim) that classically participate in distinct functions. Cytotoxic CD56dim NK cells respond to tumor targets without prior stimulation, resulting in target cell death and transient secretion of effector cytokines (e.g. IFN-γ). In contrast, immunoregulatory CD56bright NK cells secrete abundant IFN-γ and other cytokines in response to cytokine receptor stimulation, but respond minimally to tumor target-based triggering. As a result of this dichotomy, translational strategies to enhance NK cell function for cancer immunotherapy have focused exclusively on the CD56dim subset. Based upon studies in mouse NK cells, we hypothesized IL-15 priming would enhance CD56bright anti-tumor functionality. Primary human NK cells from healthy donors were purified (>95% CD56+CD3-), cultured overnight in medium alone (control) or medium with 5 ng/mL rhIL-15 (primed), washed, and assayed for anti-tumor responses. IL-15 priming significantly enhanced multiple CD56bright NK cell functional responses to the prototypical AML target cell line K562 (CD107a+: control 20% vs. primed 59%, p<0.001; IFN-γ+: 3% vs. 27%, p<0.001; TNF: 3% vs. 20%, p<0.001), as well as primary AML blasts (N=3 unique AML sample: CD107a+,7% vs. 30%, p<0.001; IFN-γ 2% vs. 14%, p<0.001; TNF: 2% vs. 22%, p<0.001). IL-15-priming of CD56bright NK cells was evident after 1 hour, and peaked after only 6 hours. In addition, flow-sorted IL-15-primed CD56bright NK cells exhibited markedly increased killing of leukemia target cells. Similar results for functional comparisons were observed using primary NK cells from AML patients. Unexpectedly, IL-15-primed CD56bright NK cell anti-leukemia responses significantly exceeded those of IL-15-primed CD56dim NK cells. Multidimensional CyTOF analyses revealed that the maturity status of CD56dim NK cells did not determine the extent to which they could be primed by IL-15. In response to IL-15, we observed selective activation of the PI3K/Akt/mTOR (4.2 fold increase CD56bright NK cells, 1.2 CD56dim NK cells, p<0.001) and Ras/Raf/MEK/ERK (1.9 fold increase CD56bright NK cells, 1.2 CD56dim NK cells, p<0.001) pathways in CD56bright NK cells. The Jak/STAT pathway was strongly activated in both CD56bright and CD56dim subsets. These data suggested a signaling mechanism for preferential CD56bright NK cell priming by IL-15. Indeed, small molecule inhibitors of PI3K/Akt/mTOR and Ras/Raf/MEK/ERK pathways abrogated the anti-tumor responses of IL-15-primed CD56bright NK cells, supporting this idea. Several NK cell effector mechanisms were enhanced in IL-15-primed CD56bright NK cells, likely contributing to their augmented anti-tumor responsiveness. These included increased cytotoxic effector protein levels (granzyme B and perforin), as well as improved conjugate formation with tumor targets. Furthermore, blocking experiments demonstrated that IL-15-primed CD56bright NK cell anti-tumor responses depended on LFA-1, CD2, and NKG2D receptor interactions with target cells. Finally, since IL-15-based therapeutics are being investigated in clinical trials, we tested whether the IL-15 super-agonist complex ALT-803 primes CD56bright NK cells in vivo in cancer patients. There was an increase in leukemia target cell-triggered degranulation (CD107a+ unprimed 7% vs. primed 30%, p<0.001) and cytokine production (IFN-γ+ 6% vs. 31%, p<0.01; TNF+ 2% vs. 20%, p<0.05) by CD56bright NK cells 24 hours post-ALT-803 administration to multiple myeloma patients, compared to unprimed, pre-therapy values. Collectively, these results suggest that CD56bright NK cells play an under-appreciated anti-tumor role in settings of abundant IL-15, such as following lymphodepleting chemotherapy, during preparation for stem cell transplantation, at sites of inflammation, or after exogenous IL-15 administration. Since CD56bright NK cells have different in vivo tissue localization (secondary lymphoid organs), distinct inhibitory, activating, and chemokine receptor expression compared to CD56dim NK cells, and are thought to be the most abundant NK cell subset when considering all human tissues, this study identifies a promising NK cell subset to harness for cancer immunotherapy. Disclosures No relevant conflicts of interest to declare.

In Vitro Expanded NK Cells Have Increased Natural Cytotoxity Receptors, TRAIL and NKG2D Expression, and Superior Tumor Cytotoxicity Compared to Short-Term IL-2 –Activated NK Cells.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 463-463 ◽  
Author(s):  
Maria Berg ◽  
Andreas Lundqvist ◽  
Dawn Betters ◽  
Richard W. Childs
Keyword(s):  
Nk Cells ◽  
Nk Cell ◽  
Ex Vivo ◽  
Natural Cytotoxicity ◽  
Short Term ◽  
Tumor Cytotoxicity ◽  
Natural Cytotoxicity Receptors

Abstract Abstract 463 IL-2 activates NK cell enhancing their capacity to lyse tumor cells. To date, most clinical studies of adoptive NK cell transfer have utilized short-term (12-16 hours) IL-2-activated NK cells. Because IL-2 alone is ineffective in expanding NK cells in vitro, the relatively low numbers of NK cells obtained for infusion following short term IL-2 activation may limit the full therapeutic impact of this approach. To obtain larger numbers of NK cells, novel ex vivo expansion protocols that utilize irradiated EBV-LCL or K562 feeder cells have recently been developed. However, concerns exist that extensive ex vivo expansion might significantly reduce the in vivo proliferative potential and long-term viability of adoptively transferred NK cells. Here we investigated for differences in phenotype, tumor cytotoxicity and in vivo persistence between short-term IL-2 activated and long-term expanded NK cells. CD56+/CD3- NK cells were isolated from normal donors by immuno-magnetic bead selection and were either activated with IL-2 (500U/ml) for 12-16 hours or were expanded in vitro over 14 days using irradiated EBV-LCL feeder cells in IL-2 containing media (500U/ml). Short-term IL-2 activated NK cells did not expand in number in contrast to EBV-LCL stimulated NK cells which expanded 400-1000 fold by culture day 14. Flow cytometry analysis revealed no differences in expression of DNAM-1, 2B4, LFA-1 or granzyme B between short-term activated and expanded NK cells. However, expanded NK cells had significantly higher expression of TRAIL, NKG2D, and the natural cytotoxicity receptors NKp30, NKp44 and NKp46 and a slight increase in KIR3DL1 and KIR2DL2/3. A 4-hour 51Cr-release assay showed expanded NK cells were significantly more cytotoxic against K562 cell compared to short-term IL-2 activated NK cells; at a 1:1 effector to target ratio, 67±6%, 26±1%, and 9±1% of K562 cells were killed by expanded, short term IL-2 activated and fresh NK cells respectively (p<0.05). Increased TRAIL expression on expanded NK cells was also associated with increased lysis of TRAIL-sensitive tumor cells (RCC tumors treated with bortezomib); at a 1:1 E:T ratio, 55±3% and 5±2% of bortezomib-treated RCC tumors were killed by expanded and short-term IL-2 activated NK cells respectively (p<0.05). We next assessed for differences in the in vivo longevity of these NK cell populations when transferred into immuno-deficient mice. Two million NK cells were labeled with a near infrared-dye (DiR; 1,1'-dioctadecyl-3,3,3',3'-tetramethylindotricarbocyanine iodide) and injected intra-peritoneal (i.p.) into CB.17 SCID-beige mice. Mice were administered IL-2 (100,000U/ml bid i.p.) for five days then underwent bioluminescent imaging using the IVIS100 system. Although FACS analysis of NK cells performed immediately prior to injection showed increased DiR fluorescent intensity in short-term IL-2 activated vs. expanded NK cells, fluorescence signal in vivo was slightly higher in the first 24-96 hours in mice that received expanded NK cells; fluorescence intensity was 5-41% (p=0.003) stronger in recipients of expanded NK cells compared to mice receiving short-term IL-2 activated NK cells. We next evaluated the in vivo anti-tumor effects of activated vs. expanded NK cells. CB.17 SCID-beige mice were injected i.p. with luciferase transduced 526 human melanoma cells three days prior to receiving an i.p. injection of short term IL-2 activated vs. expanded NK cells (+ bid i.p. IL-2). Bioluminescent imaging measuring tumor flux to calculate tumor burden and tumor doubling time showed no difference in tumor progression between both NK cell cohorts. In conclusion, these results demonstrate that ex vivo expanded NK cells are phenotypically and functionally different than short-term IL-2 activated NK cells. Expanded NK cells have increased expression of natural cytotoxicity receptors, NKG2D and TRAIL and have greater TRAIL-mediated tumor cytotoxicity compared to IL-2 activated NK cells. Importantly, despite extensive ex vivo proliferation, expanded NK cells appear maintain similar longevity in vivo as non-expanded short term IL-2 activated NK cells. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Soluble and Exosome-Bound α-Galactosylceramide Mediate Preferential Proliferation of Educated NK Cells with Increased Anti-Tumor Capacity

Cancers ◽  
2021 ◽  
Vol 13 (2) ◽  
pp. 298
Author(s):  
Arnika K. Wagner ◽  
Ulf Gehrmann ◽  
Stefanie Hiltbrunner ◽  
Valentina Carannante ◽  
Thuy T. Luu ◽  
...  
Keyword(s):  
Nk Cells ◽  
Cancer Immunotherapy ◽  
Nk Cell ◽  
Mouse Strains ◽  
Target Cells ◽  
Class I Mhc ◽  
Mhc I ◽  
Cell Responses ◽  
Missing Self

Natural killer (NK) cells can kill target cells via the recognition of stress molecules and down-regulation of major histocompatibility complex class I (MHC-I). Some NK cells are educated to recognize and kill cells that have lost their MHC-I expression, e.g., tumor or virus-infected cells. A desired property of cancer immunotherapy is, therefore, to activate educated NK cells during anti-tumor responses in vivo. We here analyze NK cell responses to α-galactosylceramide (αGC), a potent activator of invariant NKT (iNKT) cells, or to exosomes loaded with αGC. In mouse strains which express different MHC-I alleles using an extended NK cell flow cytometry panel, we show that αGC induces a biased NK cell proliferation of educated NK cells. Importantly, iNKT cell-induced activation of NK cells selectively increased in vivo missing self-responses, leading to more effective rejection of tumor cells. Exosomes from antigen-presenting cells are attractive anti-cancer therapy tools as they may induce both innate and adaptive immune responses, thereby addressing the hurdle of tumor heterogeneity. Adding αGC to antigen-loaded dendritic-cell-derived exosomes also led to an increase in missing self-responses in addition to boosted T and B cell responses. This study manifests αGC as an attractive adjuvant in cancer immunotherapy, as it increases the functional capacity of educated NK cells and enhances the innate, missing self-based antitumor response.

Preclinical Development of Edit-201, a Multigene Edited Healthy Donor NK Cell with Enhanced Anti-Tumor Function and Superior Serial Killing Activity in an Immunosuppressive Environment

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 33-33
Author(s):  
Christopher M Borges ◽  
Kevin Wasko ◽  
Jared M Nasser ◽  
Kelly Donahue ◽  
Amanda Pfautz ◽  
...  
Keyword(s):  
Cell Growth ◽  
Nk Cells ◽  
Target Cell ◽  
Nk Cell ◽  
Hematologic Malignancies ◽  
Target Cells ◽  
Publicly Traded ◽  
Killing Assay ◽  
Serial Killing

Natural killer (NK) cells distinguish tumor from healthy tissue via multiple mechanisms, including recognition of stress ligands and loss of MHC class I expression. For example, KIR mismatching enables allogenic NK cells to respond to MHC positive tumors in a similar manner to MHC negative tumors, making allogeneic NK cell therapy a promising approach for broad oncology indications. Accordingly, allogenic human HD-NK cells, including gene-modified cells, have demonstrated an impressive safety and efficacy profile when administered to patients with advanced hematologic malignancies. However, effector function of allogeneic NK cells can be diminished by the lack of functional persistence, as well as tumor-intrinsic immunosuppressive mechanisms, such as production of TGF-β. To this end, we developed a next-generation allogeneic NK cell therapy using CRISPR-Cas12a gene editing to enhance NK cell function through knockout of the genes CISH and TGFBR2. Both single and simultaneous targeting (DKO) of TGFBR2 and CISH in NK cells using CRISPR-Cas12a produced in/dels at both targets in greater than 80% of NK cells, with greater than 90% of edited NK cells viable at 72 hours post-editing. Importantly, we find that DKO NK cells do not phosphorylate the SMAD2/3 protein downstream of the TGF-b receptor complex and demonstrate increased phosphorylation of pSTAT3 and pSTAT5 upon IL-15 stimulation, consistent with protein level knockout of TGFBR2 and CISH. To determine whether DKO NK cells exhibited superior function relative to control NK cells, we first measured the ability of DKO NK cells to kill Nalm6 cells (adult B cell ALL) relative to unedited control NK cells. We find that in the presence of exogenous TGF-b, DKO NK cells demonstrate improved cytotoxicity against Nalm6 tumor targets by delaying tumor re-growth in comparison to control NK cells. To better characterize the ability of DKO NK cells to kill tumor cells, we developed an in vitro serial killing assay. In this long-duration assay, up to 30 days, control and DKO NK cells (grown in the presence of IL-15) were challenged every 48 hours with a new bolus of Nalm6 tumor targets. Both DKO and unedited NK cells control Nalm6 target cell growth for greater than 18 days (9 additions of new Nalm6 target cells), demonstrating a surprising ability for the same NK cells to serially kill new Nalm6 target cells for a prolonged period of time in vitro. We find that DKO NK cells produce higher levels of IFN-γ and TNF-α relative to control NK cells over the duration of the entire serial killing assay, suggesting that DKO NK cells can continue to produce these inflammatory cytokines even after serial killing. As many tumors, including hematologic malignancies, have high concentrations of TGF-β in their microenvironments, we next tested the ability of DKO NK cells to control the growth of Nalm6 cells in our serial killing assay in the presence of TGF-b. 10ng/mL TGF-β was added at the start of the assay as well as at each addition of new Nalm6 target cells. We observed that control NK cells fail to restrict Nalm6 target cell growth beyond 4 days (after 1 addition of new Nalm6 target cells) whereas DKO NK cells control Nalm6 target cell growth for greater than 18 days (after 9 additions of new Nalm6 target cells). Similar to the serial killing assay without TGF-b, we find that DKO NK cells produce higher concentrations of IFN-γ and TNF-α relative to control NK cells over the duration of the entire serial killing assay. Broadening our repertoire of target cells beyond B cell malignancies is now in progress, including the AML-like cell lines HL-60 and THP-1, the multiple myeloma cell line RPMI 8226, and various solid tumor targets. In summary, using CRISPR-Cas12a we demonstrated highly efficient gene editing of primary human NK cells at two unique targets designed to augment NK cell anti-tumor activity across a variety of malignancies. Most significantly, we demonstrate sustained anti-tumor serial-killing activity in the presence of the potent immunosuppressive cytokine TGF-β. Together, the increased overall effector function of CISH/TGFBR2 DKO primary human NK cells and their ability to serial kill, support their development as a potent allogeneic cell-based medicine for cancer. This potential medicine, termed EDIT-201, is being advanced to clinical study. Disclosures Borges: Editas Medicine: Current Employment, Current equity holder in publicly-traded company. Wasko:Editas Medicine: Current Employment, Current equity holder in publicly-traded company. Nasser:Editas Medicine: Current Employment, Current equity holder in publicly-traded company. Donahue:Editas Medicine: Current Employment, Current equity holder in publicly-traded company. Pfautz:Editas Medicine: Current Employment, Current equity holder in publicly-traded company. Antony:Editas Medicine: Current Employment, Current equity holder in publicly-traded company. Leary:Editas Medicine: Current Employment, Current equity holder in publicly-traded company. Sexton:Editas Medicine: Current Employment, Current equity holder in publicly-traded company. Morgan:Editas Medicine: Current Employment, Current equity holder in publicly-traded company. Wong:Editas Medicine: Current Employment, Current equity holder in publicly-traded company.

Human Cytokine-Induced Memory-Like (CIML) NK Cells Exhibit Potent Anti-Leukemia Cytotoxicity and Maintain Memory-Like Functionality After Adoptive Transfer Into Immunodeficient NOD-SCID-Gc-/- (NSG) Mice

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 4501-4501 ◽  
Author(s):  
Rizwan Romee ◽  
Jeffrey W Leong ◽  
Stephanie E Schneider ◽  
Ryan P Sullivan ◽  
Megan Cooper ◽  
...  
Keyword(s):  
Nk Cells ◽  
Adoptive Transfer ◽  
Clinical Studies ◽  
Low Dose ◽  
Nk Cell ◽  
Rest Period ◽  
Leukemia Cells ◽  
Functional Responses ◽  
Nsg Mice

Background Allogeneic NK cells are anti-leukemia immune effector lymphocytes, with evidence of activity in patients in adoptive transfer clinical studies. What is the optimal approach to prepare allogeneic NK cells for maximal effector function remains an open question for adoptive NK cell therapy. Recently described cytokine-induced memory-like (CIML) NK cells are generated following a brief (16 hour) pre-activation with a combination of IL-12+IL-15+IL-18 in both mice and humans. Following weeks or months of rest, CIML NK cells exhibit an enhanced recall IFN-g response when restimulated with K562 cells or cytokines. However, their anti-leukemic cytotoxic activity and identification of key supporting cytokines for survival and sustained functionality have not been reported. We hypothesized that CIML NK cells may have enhanced effector function against AML, providing a potential rationale for future clinical studies of CIML NK cells in AML patients. To test this hypothesis we investigated the CIML NK cell response to myeloid leukemia, including primary AML blasts, and evaluated their function following transfer into NSG mice. Methods Normal human donor NK cells (>95% purity) were cultured with low dose (1 ng/mL) IL-15 alone (control) or pre-activated with IL-12 (10 ng/ml) + IL-15 (1 ng/ml) + IL-18 (50 ng/ml) for 16 hours. After washing, the cells were cultured for 7 days in low dose IL-15 (to support survival). Following this prolonged rest period in vitro, NK cell responses were assessed after 6-hour re-stimulation with K562 leukemia cells or primary AML blasts. NK cell functional responses assessed include IFN-g production and cytotoxicity (using flow based killing assays). For adoptive transfer experiments, 5-8 x 106 human CIML NK cells or control cells were injected into sub-lethally irradiated NSG mice, and assessed for persistence, expansion and function of the adoptively transferred CIML NK or control NK cell. Additional experiments included evaluating CIML NK cells for cytokine receptor expression and effector proteins after the 7 day rest period. Results As described previously, CIML NK cells had a significantly increased IFN-g response to K562 leukemia cells (15.5 ± 3% vs. 7 ± 1%, P=0.03). CIML NK cells also exhibited a more potent IFN-g response to primary blasts from untreated, newly diagnosed AML patients (N=4 AML samples, P< 0.0001). Further, CIML NK cells demonstrated a significantly greater cytotoxic response, compared to control NK cells, upon co-incubation with K562 leukemia cells (Figure 1). Consistent with this enhanced cytotoxicity, CIML NK cells had significantly increased expression of granzyme A (P=0.005) and granzyme B (P=0.006) proteins. Further, we noted a marked induction of CD25 (IL-2Ra) after IL-12+IL-15+IL-18 pre-activation, which via the IL-2Rabg resulted in enhanced functional responses to picomolar concentrations of IL-2. This included enhanced cytotoxicity against leukemia cells, IFN-g production in response to co-stimulation with IL-12, and proliferation. To assess persistence and expansion upon adoptive transfer, NSG mice were injected with control or CIML NK cells. After 7 days (during which 75,000IU of IL-2 was injected qOD) there was a preferential expansion of the CIML NK cells in blood (11±2.6 vs. 5±1.3, P=0.01) and bone marrow (0.6±0.14 vs. 0.21±0.06, P= 0.03) in these mice as assessed by the ratio of human to mouse CD45 positive cells. Further, CIML NK cells supported in vivo in NSG mice exhibited enhanced IFN-g responses upon re-stimulation with K562 leukemia cells (10±1.5% vs. 2.5±1% IFN-g positive, P= 0.03) or IL-12+IL-15 (15±2% vs. 2±0.5%, P= 0.001). Conclusions Brief (16 hour) pre-activation with a combination of IL-12+IL-15+IL-18 results in the generation of CIML NK cells that have an enhanced IFN-g and cytotoxic response to K562 leukemia cells and primary allogeneic AML blasts. Further, CD25 is induced on CIML NK cells, which in the context of the high affinity IL-2Rabg confers selective responsiveness to low concentrations of IL-2 for proliferation, enhanced cytotoxicity, and enhanced IFN-g production. CIML NK cells may develop in vivo in NSG xenografts, and CIML NK cells appear to be selectively supported by exogenous low dose IL-2 in this context. These pre-clinical data support CIML NK cells as a novel optimization approach for NK cell adoptive immunotherapy. Disclosures: No relevant conflicts of interest to declare.

Influence Of DNA Methyltransferese Inhibitors On The Anti-Leukemic Effect Of Umbilical Cord Blood Derived NK Cells Against Acute Myeloid Leukemia

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 4496-4496
Author(s):  
Harry Dolstra ◽  
Jeannette Cany ◽  
Anniek B. van der Waart ◽  
Marleen Tordoir ◽  
Basav Nagaraj Hangalapura ◽  
...  
Keyword(s):  
Cord Blood ◽  
Nk Cells ◽  
Umbilical Cord ◽  
Umbilical Cord Blood ◽  
Nk Cell ◽  
Ex Vivo ◽  
Dnmt Inhibitors ◽  
Nsg Mice ◽  
Drug Concentrations

Natural killer (NK) cell-based immunotherapy is a promising adjuvant, relatively non-toxic therapy approach for AML. However, further improvement of NK cell-based therapy is needed to increase the clinical effect. In this regard, NK cells generated ex vivo from hematopoietic progenitor cells (HPC) may have significant clinical benefits over enriched NK cells from adult donors, including the ability to choose an appropriate killer-cell immunoglobuline-like receptor (KIR)-ligand or KIR B haplotype alloreactive donor, as well as the capacity to reach high therapeutic dosages. Previously, we reported a GMP-compliant, cytokine/heparin-based culture protocol for the ex vivo generation of highly active NK cells from CD34+ HPC isolated from cryopreserved umbilical cord blood (UCB) units. Expansion in closed, large-scale bioreactors yields a clinically relevant dose of NK cells with high purity and cytolytic activity against AML cells in vitro. Currently, a clinical phase I trial with these HPC-NK cells is ongoing in our hospital. Trafficking studies in NOD/SCID/IL2Rgnull (NSG) mice demonstrated that these HPC-NK cells migrate to the bone marrow (BM) as well as to lymphoid organs where in vivo expansion and maturation can take place. Analysis of the chemokine receptor expression profile of UCB-NK cells matched in vivo findings. Particularly, a firm proportion of UCB-NK cells functionally expressed CXCR4, what could trigger BM homing in response to its ligand CXCL12. In addition, high expression of CXCR3 and CCR6 supported the capacity of UCB-NK cells to migrate to inflamed tissues via the CXCR3/CXCL10-11 and CCR6/CCL20 axis. Importantly, a single HPC-NK cell infusion combined with supportive IL-15 administration was shown to efficiently inhibit growth of K562 leukemia cells implanted in the femur of NSG mice, resulting in significant prolongation of mice survival. Furthermore, we investigated whether modulation by the DNA methyltransferase (DNMT) inhibitors Azacytidine (Aza) and Decitabine (Deci) could further potentiate the antileukemic effect of HPC-NK cells against AML cells. In concordance with previous reports, we observed a dose-dependent effect of Aza and Deci on the growth of the AML cell lines THP1 and KG1a. In subsequent NK cell killing assays, we used clinical relevant low drug concentrations to pre-treat AML cells that did not affect HPC-NK cell viability and cytolytic function. Interestingly, increased killing of pre-treated THP1 and KG1a cells by HPC-NK cells could be observed, which was correlated with an increase in the NKG2D ligand ULBP2, the DNAM-1 ligands CD112 and CD155 as well as TRAIL-R2. Notably, maintenance of low-dose DNMT inhibitors during the KG1a/NK co-culture resulted in pronounced AML growth inhibition. To examine the effect of DNMT inhibitors in vivo, THP1.LucGFP-bearing NSG mice were treated with increasing dose of both agents, which were administered according to current standard protocols applied in humans. Data indicated that treatment with Aza or Deci at dosage equivalent in human to 12.5 and 5 mg/m2 respectively was well tolerated with minimal and/or transient weight loss, and efficiently reduced the progression of THP-1.LucGFP cells in vivo. Currently, we explore whether HPC-NK cells and DNMT inhibitors can work together to combat AML in our xenograft models. These preclinical studies may provide a rationale to investigate the possible additive and/or synergistic anti-AML effects of adoptive HPC-NK cell transfer in combination with these DNMT inhibitors in AML patients. Disclosures: Tordoir: Glycostem Therapeutics: Employment. Spanholtz:Glycostem Therapeutics: Employment.

Novel Chimeric Antigen Receptor T Cells for the Treatment of CD19-Negative Relapses Occurring after CD19-Targeted Immunotherapies

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 966-966 ◽  
Author(s):  
Marco Ruella ◽  
David Barrett ◽  
Saad S. Kenderian ◽  
Olga Shestova ◽  
Ted J. Hofmann ◽  
...  
Keyword(s):  
T Cells ◽  
Chimeric Antigen Receptor ◽  
Research Funding ◽  
Cytokine Production ◽  
Term Survival ◽  
Long Term Survival ◽  
Nsg Mice

Abstract Relapsing/refractory (r/r) B-cell Acute Lymphoblastic Leukemia (ALL) is associated with a poor prognosis in both pediatric and adult patients. Novel therapies targeting CD19 on leukemic blasts, such as anti-CD19 Chimeric Antigen Receptor T cells (CART19, CTL019) or bi-specific anti-CD19/CD3 antibodies (blinatumomab) induce significant responses in this population. However, CD19-negative relapses have been reported in 5-10% of patients following CART19 or blinatumomab therapies. This is likely due to selective pressure on leukemia sub-clones by these potent anti-CD19 agents. Hence, novel effective immunotherapies are needed in order to treat these patients. In order to identify potential additional B-ALL antigens, samples from 20 r/r patients (including two that relapsed with CD19-negative disease after treatment with CART19 therapy) were screened using a custom Quantigene RNA panel (Affymetrix) and expression on cell surface was confirmed by multiparametric flow cytometry. The IL-3 receptor α (CD123) was one of the most highly and homogeneously expressed antigens in the blasts of 16/20 r/r ALL patients, and 2/2 CD19-negative relapses. Therefore, we sought to investigate the role of CART targeting CD123 (CART123) against r/r B-ALL, focusing on treating patients with CD19-negative relapses after prior anti-CD19 directed therapy. CART123 was shown to be effective in eradicating acute myeloid leukemia in xenograft mouse models but its role in ALL has not been investigated (Gill et al, Blood, 2014). We used a 2nd generation CAR123 construct that comprised a 4-1BB (CD137) co-stimulatory domain. T cells were lentivirally transduced and expanded using anti-CD3/CD28 beads. Head-to-head in vitro comparisons between CART123 and CART19 revealed similar rates of proliferation, CD107a degranulation, cytokine production and cytotoxicity when CART were co-cultured with the CD19+CD123+ B-ALL cell line NALM-6 and with primary B-ALL blasts. For in vivo evaluation, we utilized the primary ALL model that was developed by our group (Barrett et al, Blood, 2011). In this model, primary blasts obtained from ALL patients were passaged in NOD-SCID-γ chain KO (NSG) mice, and transduced with GFP/luciferase. We injected NSG mice with 2 million primary ALL blasts i.v. (CD19+, CD123+) and after engraftment, mice were treated with CART19, CART123 or control untransduced T cells (1 million i.v.). Mice treated with control T cells succumbed quickly to disease, while mice treated with either CART19 or CART123 showed tumor eradication and long term survival (Figure 1). We then evaluated the role of CART123 in the treatment of leukemia obtained from an ALL patient that relapsed with CD19-negative disease after CART19 treatment. Both CART123 and CART19 were incubated with CD19-negative ALL blasts; CART123, but not CART19 resulted in significant degranulation, robust cytokine production, and potent cytotoxicity. To confirm these results in vivo, we established a unique model of CD19-negative B-ALL xenograft. We used primary CD19-negative blasts obtained from a pediatric patient that relapsed after CART19 therapy; CD19-negative blasts were passaged in vivo in NSG mice and stably transduced with GFP/luciferase. Importantly, the blasts retained their CD19-negative phenotype. After engraftment, mice were treated with CART19, CART123 or control T cells. CART19 and control T cells had no anti-tumor activity, while CART123 resulted in a complete eradication of the disease and long term survival in these mice (Figure 2). In conclusion, CART123 represents an important additional approach to treating B-ALL, in particular due to its activity against CD19-negative relapses. Since we have previously shown that treatment with CART123 can lead to myelosuppression, CART123 should be employed to eradicate disease prior to allogeneic transplantation. Future direction may include combining CART123 with CART19 preemptively in order to avoid CD19 antigen escapes. Figure 1 Figure 1. Figure 2 Figure 2. Disclosures Ruella: Novartis: Research Funding. Kenderian:Novartis: Research Funding. Shestova:Novartis: Research Funding. Scholler:Novartis: Research Funding. Lacey:Novartis: Research Funding. Melenhorst:Novartis: Research Funding. Nazimuddin:Novartis: Research Funding. Kalos:Novartis: CTL019 Patents & Royalties, Research Funding. Porter:Novartis: Research Funding. June:Novartis: Patents & Royalties, Research Funding. Grupp:Novartis: Consultancy, Research Funding. Gill:Novartis: Research Funding.

Novel monoclonal antibody therapeutics for metastatic castration resistant prostate cancer.

2019 ◽  
Vol 37 (15_suppl) ◽  
pp. e14222-e14222
Author(s):  
Maloy Ghosh ◽  
Kavitha Iyer Rodrigues ◽  
Sunit Maity ◽  
Sanghamitra Bhattacharjee ◽  
Yogendra Manjunath ◽  
...  
Keyword(s):  
Prostate Cancer ◽  
Immune Responses ◽  
Nk Cells ◽  
Nk Cell ◽  
Therapeutic Potential ◽  
Target Cells ◽  
Castration Resistant Prostate Cancer ◽  
Growth Reduction ◽  
Castration Resistant

e14222 Background: Therapeutic potential of innate immunity comprising Natural killer cell based targets are beginning to unravel the complexity of immune responses. NK cells recognize and induce cytotoxicity of wide range of target cells, such as, tumor cells without prior antigen sensitization. In this study, we have studied Lectin-like transcript 1 (LLT1), a member of the C-type lectin super family, is expressed on target cells and various immune cells. LLT1 isoform 1, is known to interact with CD161, a critical receptor on NK cells. CD161 is expressed on most of human NK cells, NK-T cells, γδ T cells and so on. Tumors exploit the CD161- LLT1 interaction to evade host defense mechanism (“DO NOT KILL” signal); indicating LLT1 as an attractive immunotherapeutic strategy. Methods: Prostate cancer cell lines and other tumor cell lines were used to evaluate novel anti LLT1 antibodies for therapeutic potential - IFNγ production assays and tumor cell death assays were carried out. In vivo efficacy of these antibodies were established using PC3 xenograft in humanized mouse model (HuNOG-EXL). Results: Human androgen independent prostate cancer cell line, PC3 was studied for LLT1 expression and interactions with immune cells, to understand role of LLT1 in metastatic castration resistant prostate cancer (mCRPC). Overexpression of LLT1 on tumor cells was influenced by cytokines and various TLRs. Inhibition of CD161-LLT1 interaction with novel anti LLT1 antibodies leads to IFNγ production and consequent NK cell mediated cytotoxicity – hall mark of anti-tumor responses. Disruption of LLT1 - CD161 innate immunity axis with anti LLT1 antibody releases the break on NK cell cytotoxicity and hence, established a new therapeutic option. PC3 xenograft on HuNOG mouse revealed in vivo efficacy of LLT1 antibody. Significant tumor growth reduction was observed with specific anti LLT1 antibodies alone and in combination with check point antibodies. Thus, synergistic tumor growth reduction was established by combinatorial application of anti LLT1 antibody and PD1/PDL1 axis inhibitors. Conclusions: PC3 xenograft study and other results point to therapeutic opportunities in metastatic castration resistant prostate cancer, a disease condition which needs improved patient outcomes. The ligation of CD161/LLT1 will serve as a new immuno-oncology pair regulating innate and adaptive immune responses; novel human antibodies against LLT1 described here will bring therapeutic benefit to patients in need.

p38 MAPK activation controls the TLR3-mediated up-regulation of cytotoxicity and cytokine production in human NK cells

Blood ◽  
2004 ◽  
Vol 104 (13) ◽  
pp. 4157-4164 ◽  
Author(s):  
Simona Pisegna ◽  
Gianluca Pirozzi ◽  
Mario Piccoli ◽  
Luigi Frati ◽  
Angela Santoni ◽  
...  
Keyword(s):  
Nk Cells ◽  
P38 Mapk ◽  
Cytokine Production ◽  
Viral Infections ◽  
Nk Cell ◽  
Mitogen Activated Protein Kinase ◽  
Poly I:C ◽  
Central Component ◽  
Polycytidylic Acid

Abstract Natural killer (NK) cells are a component of the innate immunity against viral infections through their rapid cytotoxic activity and cytokine production. Although the synthetic double-stranded (ds) RNA polyinosinic-polycytidylic acid (poly I:C), a mimic of a common product of viral infections, is known to rapidly up-regulate their in vivo functions, NK cell ability to directly respond to dsRNA is still mostly unknown. Our results show that treatment with poly I:C significantly up-regulates both natural and CD16-mediated cytotoxicity of highly purified human NK cells. Poly I:C also induces the novel capability of producing CXCL10 chemokine in human NK cells and synergistically enhances interferon-γ (IFN-γ) production induced by either adaptive or innate cytokines. In accordance with the expression of Toll-like receptor-3 (TLR3) and of TRIF/TICAM-1 adaptor, poly I:C stimulation induces the activation of interferon regulatory factor-3 (IRF-3) transcription factor and of p38 mitogen-activated protein kinase (MAPK) in human NK cells. Finally, we demonstrate that p38 MAPK activity is required for the dsRNA-dependent enhancement of cytotoxicity and CXCL10 production. The occurrence of dsRNA-induced signaling and functional events closely correlates with the TLR3 mRNAprofile in different NK cell populations. Taken together, these data identify p38 as a central component of NK cell ability to directly respond to dsRNA pathogen-associated molecular pattern (PAMP).

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