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

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.

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The JAK Inhibitor Ruxolitinib Substantially Affects NK Cells in MPN Patients

Blood ◽

10.1182/blood.v124.21.3169.3169 ◽

2014 ◽

Vol 124 (21) ◽

pp. 3169-3169

Author(s):

Kathrin Schönberg ◽

Janna Rudolph ◽

Maria Vonnahme ◽

Isabelle Cornez ◽

Sowmya Parampalli Yajnanarayana ◽

...

Keyword(s):

Nk Cells ◽

Research Funding ◽

Nk Cell ◽

Receptor Expression ◽

Target Cells ◽

Jak Inhibitor ◽

Advisory Committees ◽

Nk Cell Differentiation

Abstract Introduction: Ruxolitinib (INCB018424) is the first JAK inhibitor approved for treatment of myelofibrosis (MF). Ruxolitinib-induced reduction of splenomegaly and symptoms control is linked to a substantial suppression of MF-associated circulating pro-inflammatory and pro-angiogenic cytokines. However, an increased rate of infections in ruxolitinib-exposed patients with MF was recently described. Natural killer (NK) cells are innate immune effector cells eliminating malignant or virus-infected cells. Thus, the aim of this project was to define in more detail the impact of JAK inhibition on NK cell biology both in vitro and in vivo. Methods: 28 patients with myeloproliferative neoplasms (MPN) with or without ruxolitinib therapy and 12 healthy donors were analyzed for NK cell frequency, NK receptor expression and function. Phenotypic and functional NK cell markers (e.g. CD11b, CD27, KIR, NKG2A, NKG2D, NKp46, CD16, granzyme B, and perforin) were analyzed by FACS. NK cell function was evaluated by classical killing assays upon stimulation with MHC class I-deficient target cells K562. Finally, a set of additional in vitro experiments (e.g. analysis of lytic synapse formation by FACS and confocal microscopy) were performed to define in more detail the characteristics and potential mechanisms of ruxolitinib-induced NK cell dysfunction. Results: In addition to our recent finding that ruxolitinib induces NK cell dysfunction in vitro (e.g. reduced killing, degranulation and IFN-γ production), we here demonstrate that NK cell proliferation and cytokine-induced receptor expression as well as cytokine signalling are drastically impaired by ruxolitinib. Interestingly, reduced killing is at least in part due to a reduced capacity to form a mature lytic synapse with target cells. The significance of the in vitrofindings is underscored by a dramatically reduced proportion and absolute number of NK cells in ruxolitinib-treated MPN patients when compared to treatment-naïve patients or to healthy controls (mean percentage of NK cell frequency: ruxolitinib-naïve MPN patients 12.63% ±1.81; healthy donors 13.51% ±1.44; ruxolitinib-treated patients 5.47% ±1.27). A systematic analysis of NK cell receptor expression revealed that the reduction of NK cells in ruxolitinib-exposed individuals is most likely due to an impaired NK cell differentiation and maturation process, as reflected by a significantly increased ratio of immature to mature NK cells. Finally, the endogenous functional NK cell defect in MPN is further aggravated by intake of the JAK inhibitor ruxolitinib. Conclusion: We here provide compelling in vitro and in vivo evidence that inhibition of the JAK/STAT-pathway by ruxolitinib exerts substantial effects on the NK cell compartment in MPN patients due to the inhibition of NK cell differentiation and NK cell key functions. Our data may help to better understand the increased rate of severe infections and complement recent reports on ruxolitinib-induced immune dysfunction. Disclosures Koschmieder: Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Travel, Accomodation, Expenses Other. Brümmendorf:Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding. Wolf:Novartis: Consultancy, Honoraria, Research Funding, Travel and Accommodation Other.

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CD58 Immunobiology at a Glance

Frontiers in Immunology ◽

10.3389/fimmu.2021.705260 ◽

2021 ◽

Vol 12 ◽

Author(s):

Yalu Zhang ◽

Qiaofei Liu ◽

Sen Yang ◽

Quan Liao

Keyword(s):

Nk Cells ◽

Intracellular Signaling ◽

Inflammatory Responses ◽

Nk Cell ◽

Costimulatory Molecule ◽

Soluble Form ◽

Target Cells ◽

Lymphocyte Function

The glycoprotein CD58, also known as lymphocyte-function antigen 3 (LFA-3), is a costimulatory receptor distributed on a broad range of human tissue cells. Its natural ligand CD2 is primarily expressed on the surface of T/NK cells. The CD2-CD58 interaction is an important component of the immunological synapse (IS) that induces activation and proliferation of T/NK cells and triggers a series of intracellular signaling in T/NK cells and target cells, respectively, in addition to promoting cell adhesion and recognition. Furthermore, a soluble form of CD58 (sCD58) is also present in cellular supernatant in vitro and in local tissues in vivo. The sCD58 is involved in T/NK cell-mediated immune responses as an immunosuppressive factor by affecting CD2-CD58 interaction. Altered accumulation of sCD58 may lead to immunosuppression of T/NK cells in the tumor microenvironment, allowing sCD58 as a novel immunotherapeutic target. Recently, the crucial roles of costimulatory molecule CD58 in immunomodulation seem to be reattracting the interests of investigators. In particular, the CD2-CD58 interaction is involved in the regulation of antiviral responses, inflammatory responses in autoimmune diseases, immune rejection of transplantation, and immune evasion of tumor cells. In this review, we provide a comprehensive summary of CD58 immunobiology.

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MicroRNA-Deficient Murine NK Cells Exhibit Impaired Development and Survival but Enhanced IFN-γ Production In Vitro and In Vivo

Blood ◽

10.1182/blood.v118.21.357.357 ◽

2011 ◽

Vol 118 (21) ◽

pp. 357-357 ◽

Cited By ~ 1

Author(s):

Ryan P Sullivan ◽

Jeffrey W Leong ◽

Stephanie E Schneider ◽

Catherine R Keppel ◽

Elizabeth Germino ◽

...

Keyword(s):

Nk Cells ◽

Cell Biology ◽

Nk Cell ◽

Cell Receptor ◽

Cell Development ◽

Target Cells ◽

Ifn Γ ◽

Nk Cell Receptor

Abstract Abstract 357 NK cells are innate immune lymphocytes important for early host defense against infectious pathogens and malignant transformation. MicroRNAs (miRNAs) are small regulatory RNAs that control a wide variety of cellular processes by specific targeting of mRNA 3'UTRs. The Dicer1 gene encodes a conserved enzyme essential for miRNA processing, and Dicer1 deficiency leads to a global defect in miRNA biogenesis. While miRNA expression and regulation of adaptive T and B lymphocytes are well established, their role in the regulation of NK cell biology remains unclear. We postulated that miRNAs serve an essential role in orchestrating NK cell development and activation. To test this hypothesis, we combined lymphocyte-restricted hCD2-Cre transgenic, Rosa26-YFP-Cre-reporter, and Dicer1 ‘floxed' mice. In this model, 25–50% of Dicer1 wt/wt NK cells are YFP+ marking expression of Cre. As expected, YFP+ NK cells from Dicer1 fl/fl and fl/wt mice were confirmed to excise Dicer1, and exhibit decreased total miRNA content based on Nanostring profiling and real-time qPCR (Dicer1 fl/fl: P<0.001, fl/wt: P<0.01). MiRNA-deficient Dicer1 fl/fl mice exhibited reduced YFP+ NK percentages (spleen Dicer1 fl/fl: 14±4%, fl/wt: 35±7%, wt/wt 36±7%, P<0.001) as well as reduced absolute numbers of YFP+ NK cells [spleen Dicer1 fl/fl: 3.4±0.6×10E5, fl/wt: 6.3±1.7×10E5, wt/wt 6.1±.99×10E5, P<0.01]. In addition, Dicer1 fl/fl mice had reductions NK cell precursors in the BM (stage 2–3 NK precursors mean decrease 70±14% in Dicer1 fl/fl compared to wt/wt, P <0.01). Further, Dicer1 fl/fl NK cells exhibited reduced survival ex vivo when cultured in medium (P<0.01), low dose- (P<0.01), or high dose-IL-15 (P<0.01). These data collectively indicate that Dicer1-dependent miRNAs regulate NK cell development and homeostasis, and the net effect of miRNA loss is impaired NK development and/or survival. However, in our model Dicer1-deficient mature NK cells exhibited enhanced functionality; a finding that contrasts to less NK selective miRNA-deficient NK cell models (Bezman et al. J Immunol 185:3835, 2010). Degranulation (CD107a+, a surrogate for cytotoxicity) was enhanced in vitro in response to YAC-1 tumor target cells (P<0.05) and activating NK cell receptor ligation (P<0.001). This was unlikely due to alteration in activating NK cell receptor expression since the surface density of NKG2D and NKp46 were not affected by miRNA deficiency. Moreover, interferon-gamma (IFN-γ) production was enhanced in vitro in miRNA-deficient NK cells in response to IL-12+IL-15 (P<0.01), YAC-1 tumor target cells (P<0.01), and activating NK cell receptor ligation (P<0.001). Further, evaluation of NK cells 36 hours after infection with MCMV resulted in significantly increased IFN-γ production (% NK YFP+IFN-γ+) in Dicer1 fl/fl (64± 4.9%) vs. fl/wt (52±11%, p <0.01) or vs. wt/wt (41±6%, p <0.001) in vivo. MiRs-15/16 were identified as abundant miRNAs in NK cells that had reduced expression in Dicer1 fl/fl NK cells, and are predicted to target the murine IFN-γ 3'UTR. This targeting was validated in vitro, by transfecting 293T cells with miRNA-15/16 or control over-expression vectors and a sensor plasmid that places luciferase under the control of the murine IFN-γ 3'UTR (34% decrease, P<0.01). Moreover, the targeting was direct, since miR-15/16 targeting of IFN-γ was abrogated after mutation of two predicted binding sites in the IFN-γ 3'UTR. These data indicated that miR-15/16 may regulate IFN-γ translation by resting NK cells. Thus, our study suggests that the function of miRNAs in NK cell biology is complex, with an important role in NK cell development, survival and/or homeostasis, while tempering peripheral NK cell activation. Further study of individual miRNAs in an NK cell specific fashion will provide insight into these complex miRNA regulatory effects in NK cell development/survival and effector function. Disclosures: No relevant conflicts of interest to declare.

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Exploitation of natural killer cells for the treatment of acute leukemia

Blood ◽

10.1182/blood-2015-12-629055 ◽

2016 ◽

Vol 127 (26) ◽

pp. 3341-3349 ◽

Cited By ~ 58

Author(s):

Rupert Handgretinger ◽

Peter Lang ◽

Maya C. André

Keyword(s):

Cell Therapy ◽

Nk Cells ◽

Natural Killer ◽

Adoptive Transfer ◽

Nk Cell ◽

Lymphoblastic Leukemia ◽

Leukemia Cell ◽

Target Cells

Abstract Natural killer (NK) cells play an important role in surveillance and elimination of malignant cells. Their spontaneous cytotoxicity was first demonstrated in vitro against leukemia cell lines, and NK cells might play a crucial role in the therapy of leukemia. NK cell activity is controlled by an array of germ line–encoded activating and inhibitory receptors, as well as modulating coreceptors. This biologic feature can be exploited in allogeneic cell therapy, and the recognition of “missing-self” on target cells is crucial for promoting NK cell–mediated graft-versus-leukemia effects. In this regard, NK cells that express an inhibitory killer immunoglobulin-like receptor (iKIR) for which the respective major histocompatibility complex class I ligand is absent on leukemic target cells can exert alloreactivity in vitro and in vivo. Several models regarding potential donor–patient constellations have been described that have demonstrated the clinical benefit of such alloreactivity of the donor-derived NK cell system in patients with adult acute myeloid leukemia and pediatric B-cell precursor acute lymphoblastic leukemia after allogeneic stem cell transplantation. Moreover, adoptive transfer of mature allogeneic NK cells in the nontransplant or transplant setting has been shown to be safe and feasible, whereas its effectivity needs further evaluation. NK cell therapy can be further improved by optimal donor selection based on phenotypic and genotypic properties, by adoptive transfer of NK cells with ex vivo or in vivo cytokine stimulation, by the use of antibodies to induce antibody-dependent cellular cytotoxicity or to block iKIRs, or by transduction of chimeric antigen receptors.

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

Blood ◽

10.1182/blood.v128.22.550.550 ◽

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.

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In vitro education of human natural killer cells by KIR3DL1

Life Science Alliance ◽

10.26508/lsa.201900434 ◽

2019 ◽

Vol 2 (6) ◽

pp. e201900434

Author(s):

Jason Pugh ◽

Neda Nemat-Gorgani ◽

Zakia Djaoud ◽

Lisbeth A Guethlein ◽

Paul J Norman ◽

...

Keyword(s):

Nk Cells ◽

Nk Cell ◽

Hla Class I ◽

Surface Expression ◽

Class I ◽

Target Cells ◽

Human Natural Killer Cells ◽

Basic Protocol

During development, NK cells are “educated” to respond aggressively to cells with low surface expression of HLA class I, a hallmark of malignant and infected cells. The mechanism of education involves interactions between inhibitory killer immunoglobulin–like receptors (KIRs) and specific HLA epitopes, but the details of this process are unknown. Because of the genetic diversity of HLA class I genes, most people have NK cells that are incompletely educated, representing an untapped source of human immunity. We demonstrate how mature peripheral KIR3DL1+ human NK cells can be educated in vitro. To accomplish this, we trained NK cells expressing the inhibitory KIR3DL1 receptor by co-culturing them with target cells that expressed its ligand, Bw4+HLA-B. After this training, KIR3DL1+ NK cells increased their inflammatory and lytic responses toward target cells lacking Bw4+HLA-B, as though they had been educated in vivo. By varying the conditions of this basic protocol, we provide mechanistic and translational insights into the process NK cell education.

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

Blood ◽

10.1182/blood-2020-140848 ◽

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.

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Depletion of the C3 Component of Complement Enhances the Ability of Rituximab-Coated Target Cells to Activate Human NK Cells and Improves the Efficacy of Monoclonal Antibody Therapy in An in Vivo Model

Blood ◽

10.1182/blood.v112.11.3620.3620 ◽

2008 ◽

Vol 112 (11) ◽

pp. 3620-3620

Author(s):

Siao- Yi Wang ◽

Jeffrey Cagley ◽

David Fritzinger ◽

Carl-Wilhelm Vogel ◽

William St. John ◽

...

Keyword(s):

Nk Cells ◽

Median Survival ◽

Nk Cell ◽

Antibody Therapy ◽

Malignant Ascites ◽

In Vivo Model ◽

Target Cells ◽

Lymphoma Cells

Abstract Antibody-dependent cellular cytotoxicity (ADCC) is felt to play an important role in mediating the anti-tumor effects of Rituximab (R). We previously reported that C3b deposition on R-coated target cells interferes with the binding of the Fc portion of R to NK cell CD16. This interaction inhibits the activation of NK cells and NK cell-mediated ADCC of 51Cr-labeled R-coated target cells. The current studies were designed to determine whether C3 depletion enhances the ability of mAb-coated targets to activate NK cells in vitro and improves mAb therapy in vivo. Normal human serum inhibited the ability of R-coated lymphoma cells to activate NK cells as previously reported. NK activation was increased when serum was pre-incubated with cobra venom factor (CVF) to deplete C3. Similar results were found when non-malignant ascites or transudative pleural fluid, as a surrogate for extravascular fluid, was used as the source of complement. For in vivo analysis, we utilized a syngeneic, immunocompetent murine model in which ADCC has been previously demonstrated to be a key mechanism of action. CVF or a human C3 derivative with CVF-like functions (HC3-1496) was used to deplete C3 in vivo. In this model, C3H/HeN mice were inoculated with murine 38C13 lymphoma cells (day 0) and treated with 2 doses of CVF or HC3-1496 (day 3 and day 5). Four hours after the initial dose of CVF or HC3-1496, mice were treated with a single dose of an anti-lymphoma mAb directed against the 38C13 idiotype (MS11G6). Untreated mice all developed tumor and died with a median survival of 28 days. All mice treated with mAb alone eventually developed tumor and died with a median survival of 42 days. Survival following treatment with CVF plus mAb was superior to that of mAb alone (Fig 1, p=0.0312) with 50% of mice remaining tumor free. Survival following treatment with HC3-1496 plus mAb was also superior to that of mAb alone (Fig 2, p=0.0002) with 80% of mice remaining tumor free. In summary, depletion of the C3 component of complement enhanced the ability of R-coated target cells to activate human NK cells, and improved the efficacy of mAb therapy in an in vivo model of lymphoma. Furthermore, these studies suggest the inhibitory effects of complement on NK activation and ADCC may be seen in the extravascular compartment such as within involved lymph nodes. We conclude that depletion of complement through use of agents such as CVF or HC3-1496 could be considered as an approach to enhancing the efficacy of R therapy. Figure Figure Figure Figure

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A Bispecific Protein Targeting the NKG2D Receptor on Natural Killer Cells: In Vitro and In Vivo activity of ULBP2-CEA

Blood ◽

10.1182/blood.v116.21.2095.2095 ◽

2010 ◽

Vol 116 (21) ◽

pp. 2095-2095

Author(s):

Marie Madlener ◽

Achim Rothe ◽

Ron Jachimowicz ◽

Katrin S. Reiners ◽

Sampurna Chatterjee ◽

...

Keyword(s):

Nk Cells ◽

Natural Killer ◽

Nk Cell ◽

Carcinoma Cells ◽

Target Cells ◽

Colon Carcinoma Cells ◽

Syngeneic Mouse Model ◽

Nkg2d Receptor

Abstract Abstract 2095 Natural killer (NK) cells are effector lymphocytes of the innate immune system. NK cell activity is tightly regulated by inhibitory and activating surface receptors to ensure that NK cells recognize and eliminate target cells, whereas healthy cells escape from the NK cell-mediated immune surveillance. The inability of the immune system to recognize and kill malignant target cells has been partly attributed to the ineffective activation of NK cells. It is well accepted, that the activating NK receptor NKG2D (natural killer group 2 member D) plays an important role in tumor rejection. The Immunoligand ULBP2-CEA is a recombinant bispecific protein designed to enhance NK cell-mediated lysis of CEA-expressing target cells. Consisting of the NKG2D-ligand ULBP2 (UL16 binding protein 2) and an anti-CEA single chain antibody, this construct provides the ability of cross-linking NK cells and CEA-expressing cells for specific tumor targeting. CEA (carcinoembryonic antigen) is frequently overexpressed in colorectal and other carcinomas. The binding and specificity of ULBP2-CEA fusion protein was tested in FACS analysis. Transfected human CEA+ murine colon carcinoma cells (MC38-CEA) were incubated with ULBP2-CEA and recombinant NKG2D receptor (NKG2DR). The protein was able to bind both, tumor cells and recombinant NKG2DR simultaneously. No specific cell binding was detectable in control experiments using fusion constructs with another binding moiety (e.g. ULBP2-BB4) or soluble NKG2DR alone. In cytotoxicity assays ULBP2-CEA enhances the susceptibility to NK cell mediated lysis of CEA-expressing cells. CEA+ human colon carcinoma cells (LS174T) were incubated with NK cells isolated from healthy donors in the presence or absence of ULBP2-CEA. The increase of cell lysis was significant at all effector-target ratios compared to the controls demonstrating specific targeted in vitro efficacy of ULBP2-CEA. In order to investigate anti-tumor activity in vivo we use a syngeneic mouse model. MC38-CEA cells were implanted subcutaneously in CEA transgenic C57BL/6 mice and tumor growth was assessed using in vivo bioluminescence imaging (BLI). First results on the in vivo efficacy of ULBP-CEA and effects on the activation status of splenic and peripheral blood NK and T cells upon treatment will be discussed. This syngeneic mouse model is a useful tool to study efficacy and mode of action of ULBP2-CEA in vivo. Disclosures: No relevant conflicts of interest to declare.

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Depletion of the C3 component of complement enhances the ability of rituximab-coated target cells to activate human NK cells and improves the efficacy of monoclonal antibody therapy in an in vivo model

Blood ◽

10.1182/blood-2009-01-200469 ◽

2009 ◽

Vol 114 (26) ◽

pp. 5322-5330 ◽

Cited By ~ 93

Author(s):

Siao-Yi Wang ◽

Suresh Veeramani ◽

Emilian Racila ◽

Jeffrey Cagley ◽

David C. Fritzinger ◽

...

Keyword(s):

Monoclonal Antibody ◽

Nk Cells ◽

Cell Activation ◽

Nk Cell ◽

Antibody Therapy ◽

Inhibitory Effect ◽

In Vivo Model ◽

Target Cells

Abstract Growing evidence indicates antibody-dependent cellular cytotoxicity (ADCC) contributes to the clinical response to monoclonal antibody (mAb) therapy of lymphoma. Recent in vitro analysis suggests C3b can inhibit mAb-induced natural killer (NK)–cell activation and ADCC. Further studies were conducted to assess the effect of C3 depletion on mAb-induced NK activation and therapy of lymphoma. Normal human serum inhibited the ability of rituximab-coated lymphoma cells to activate NK cells as previously reported. Serum did not inhibit NK-cell activation when it was preincubated with cobra venom factor (CVF) to deplete C3. Similar results were found when transudative pleural fluid or nonmalignant ascites was used as surrogates for extravascular fluid, suggesting the inhibitory effect of complement may be present in the extravascular compartment, in which many malignant lymphocytes reside. In vivo, C3 was depleted before mAb treatment in a syngeneic murine model of lymphoma. Survival of lymphoma-bearing mice after treatment with CVF plus mAb and with a human C3 derivative with CVF-like functions (HC3-1496) plus mAb was both superior to that of mAb alone. These studies show that complement depletion enhances NK-cell activation induced by rituximab-coated target cells and improves the efficacy of mAb therapy in a murine lymphoma model.

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