Activation of T- and NK- Cells through CD19xCD3 and CD19xCD16A Bispecific TandAb Antibodies in Patients with B-Cell Non-Hodgkin's Lymphoma.

Abstract Abstract 1950 Poster Board I-973 Purpose: Bispecific, tetravalent.antibodies (TandAbs) directed against the B-cell-surface marker CD19 and activating receptors on T or NK cells (CD19xCD3 or CD19xCD16b) have shown promising effects in vitro and in preclinical studies. The present study examines their effects on the cytotoxic responses of viable T and NK cells isolated from patients with B-cell Non Hodgkin Lymphoma (NHL). Methods: Peripheral Blood MNCs were obtained from 30 patients with B-NHL (High grade=6, low grade=24) after successful prior therapy and from 17 healthy donors (HD) and were enriched for NK cells by immunomagnetic separation with CD56-microbeads, the other one was enriched for T cells. After overnight stimulation (RPMI-1640 with 10% FCS, 1% Pen/Strep and human recombinant IL-2 (Novartis) (NK: 1000U/ml; T: 100U/ml) cells were stained with CD107a, a surface marker indicating cytotoxicity. Cells were exposed to K562 alone, to Raji cells alone and finally to the TandAbs CD19xCD3 (T cells) or CD19xCD16A (NK cells) (conc. 1μg/ml). Using FACS analysis we determined the percent fraction of activated CD3 or CD56 cells being positive for CD107a. Results: To look for functional NK cells and T cells of HD and NHL patients first we exposed the cells to the K562 cell line. Both groups, HD and NHL patients expressed CD107a similarly (NK: 27.7±12.0% vs. 28.5±14.6%; T: 9.75±7.1% vs. 8.1±5.7%), indicating functional NK and T cells. To observe if NK or T cells would upregulate CD107a expression when exposed to lymphoma cells alone we performed experiments with Raji cells and measured the CD107a response. NK cells of both, patients and HD responded with similar levels of CD107a expression. T-cells of patients showed more cytotoxic activity towards Raji cells than observed in HD (3.1±8.4% vs. 0.1±0.2%). Next we added the CD19xCD3 or the CD19xCD16 TandAb to T/NK cells in the presence of Raji cells. In the patients′ NK-cell-population the fraction of activated cells increased from 10.7% (Raji cells alone) to 30.7±19.2% when TandAb was added. The mean reaction in the group of HD was increased from 9.2% up to 27.7%. CD107a expression of T cells from NHL patients increased from 3.1% when exposed to Raji alone up to 21.3±14.3% when TandAb was added which was comparable to the results of HD (19.6%±11.4%). Conclusion: Patients with NHL have functional intact effector cells which show cytotoxic activity in response to TandAbs in presence of lymphoma cells similar to that of HD. These TandAb antibodies are therefore suitable for patients with B-cell NHL as potential maintenance therapy. Disclosures: Reusch: Affimed: Employment, Patents & Royalties. Little:Affimed: Employment, Equity Ownership, Patents & Royalties.

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Optimizing NK-92 Serial Killers: Gamma Irradiation, CD95/FasLigation, And NK Or LAK Attack Limit Cytotoxic Efficacy

10.21203/rs.3.rs-1193866/v1 ◽

2021 ◽

Author(s):

Lydia T Navarrete-Galvan ◽

Michael Guglielmo ◽

Judith Cruz Amaya ◽

Julie Smith-Gagen ◽

Vincent C. Lombardi ◽

...

Keyword(s):

Gamma Irradiation ◽

Cytotoxic Activity ◽

Nk Cells ◽

Nk Cell ◽

Death Receptor ◽

Serial Killers ◽

Raji Cells ◽

Serial Killing ◽

Genetic Deletion ◽

High Cytotoxic Activity

Abstract Background: The NK cell line NK-92 and its genetically modified variants are receiving attention as immunotherapies to treat a range of malignancies. However, since NK-92 cells are themselves tumors, they require irradiation prior to transfer and are potentially susceptible to attack by patients’ immune systems. Here, we investigated NK-92 cell-mediated serial killing for the effects of gamma-irradiation and ligation of the death receptor Fas (CD95), and NK-92 cell susceptibility to attack by activated primary blood NK cells. Methods: To evaluate serial killing, we used 51 Cr-release assays with low NK-92 effector cell to target Raji, Daudi or K562 tumor cell (E:T) ratios to determine killing frequencies at 2-, 4-, 6-, and 8-hours. Results: NK-92 cells were able to kill up to 14 Raji cells per NK-92 cell in eight hours. NK-92 cells retained high cytotoxic activity immediately after irradiation with 10 Gy but the cells surviving irradiation lost >50% activity one day after irradiation. Despite high expression of CD95, NK-92 cells maintained their viability following overnight Fas/CD95-ligation but lost some cytotoxic activity. However, one day after irradiation, NK-92 cells were more susceptible to Fas ligation, resulting in decreased cytotoxic activity of the cells surviving irradiation. Irradiated NK-92 cells were also susceptible to killing by both unstimulated and IL-2 activated primary NK cells (LAK). In contrast, non-irradiated NK-92 cells were more resistant to attack by NK and LAK cells. Conclusions: Irradiation is deleterious to both the survival and cytotoxicity mediated by NK-92 cells and renders the NK-92 cells susceptible to Fas-initiated death and death initiated by primary blood NK cells. Therefore, replacement of irradiation as an antiproliferative pretreatment and genetic deletion of Fas and/or NK activation ligands from adoptively transferred cell lines are indicated as new approaches to increase therapeutic efficacy.

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Mechanical Regulation of the Cytotoxic Activity of Natural Killer Cells

10.1101/2020.03.02.972984 ◽

2020 ◽

Author(s):

L. Mordechay ◽

G. Le Saux ◽

A. Edri ◽

U. Hadad ◽

A. Porgador ◽

...

Keyword(s):

T Cells ◽

B Cells ◽

Cytotoxic Activity ◽

Nk Cells ◽

Natural Killer ◽

Nk Cell ◽

Activation Mechanism ◽

Catch Bond ◽

Immune Activity ◽

The Third

AbstractMechanosensing has been recently explored for T cells and B cells and is believed to be part of their activation mechanism. Here, we explore the mechanosensing of the third type of lymphocytes – Natural Killer (NK) cells, by showing that they modulate their immune activity in response to changes in the stiffness of a stimulating surface. Interestingly, we found that this immune response is bell-shaped, and peaks for a stiffness of a few hundreds of kPa. This bell-shape behavior was observed only for surfaces functionalized with the activating ligand MHC class I polypeptide-related sequence A (MICA), but not for control surfaces lacking immunoactive functionalities. We found that stiffness does not affect uniformly all cells but increases the size of a little group of extra-active cells, which in turn contribute to the overall activation effect of the entire cell population. We further imaged the clustering of costimulatory adapter protein DAP10 on NK cell membrane and found that it shows the same bell –shape dependence to surface stiffness. Based on these findings, we propose a catch-bond-based model for the mechanoregulation of NK cell cytotoxic activity, through interaction of NKG2D activating receptors with MICA. Our findings reveal what seems to be “the tip of iceberg” of mechanosensation of NK cells, and provides an important insight on the mechanism of their immune signaling.Statement of SignificanceThe mechanical sensing of immune lymphocytes was recently demonstrated for T cells and B cells, but not for the third type of lymphocytes – Natural Killer (NK) cells. Interestingly, previous reports on lymphocyte mechanosensing were controversial, and showed either positive or negative changes in their immune activity with environmental stiffness, depending on the stiffness range. In this paper, we directly demonstrated that NK cells modulate their response with the stiffness of the stimulating surface, and this modulation has a bell-shape trend. We found that there is a strong correlation between the response to stiffness and clustering of adaptor proteins. Upon this correlation, we proposed a mechanosensing model based on the catch-bond nature of activating ligand-receptor complexes in NK cells.

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Lentiviral Transduction of Ex Vivo Expanded Natural Killer Cells with a CD19 Chimeric Antigen Receptor Induces Cytotoxicity against Resistant B Cell Malignancies

Blood ◽

10.1182/blood.v112.11.3540.3540 ◽

2008 ◽

Vol 112 (11) ◽

pp. 3540-3540

Author(s):

Muthalagu Ramanathan ◽

Su Su ◽

Andreas Lundqvist ◽

Maria Berg ◽

Aleah Smith ◽

...

Keyword(s):

T Cells ◽

B Cell ◽

Nk Cells ◽

Nk Cell ◽

Ex Vivo ◽

Cell Cytotoxicity ◽

Gfp Expression ◽

B Cell Malignancies ◽

Activated T Cells ◽

Nk Cell Cytotoxicity

Abstract NK cells play an important role in innate immunity against tumors and viral infection. NK cell cytotoxicity is suppressed by self-HLA molecules that bind and activate inhibitory killer immunoglobulin like receptors (KIRs). Expression of a CD19 chimeric receptor on NK cells could induce target specific activating signals that overcome KIR-mediated inhibition, enhancing autologous NK cell cytotoxicity against B-cell malignancies. Although HIV-1 based lentiviral vectors (LVs) have been used to efficiently transfer genes into human T-cells, little data exists on the use of LV vectors to transduce NK cells. In this study, we designed a HIV-based LV vector encoding both a CD19 chimeric antigen receptor (CAR) and green fluorescence protein (GFP) transgenes controlled by a MSCV-LTR promoter (CD19CAR LV vector) to transduce CD3−CD56+ ex vivo expanded human NK cells. The CAR consists of a single chain Fv portion of a mouse mAb against human CD19 fused to the signaling intracellular domain of a CD3 zeta subunit. CD3−CD56+NK cells were expanded ex vivo using irradiated EBV-LCL feeder cells and IL-2 containing media for 7 to 10 days. NK92 cells or expanded NK cells underwent 2 rounds of transduction with the CD19CAR LV vector in the presence of protamine sulfate using retronectin-coated plates. GFP expression measured by flow cytometry 3–4 days following LV transduction was used to assess transduction efficiencies (TE). GFP expression was detected in a mean 41% (range 27–56%) of NK92 cells and a mean 15% (range 6–40%) of ex vivo expanded NK cells. NK cell viability assessed up to 1 week following LV transduction was similar to non transduced NK cells. Following transduction, NK cells continued to expand in culture similar to non-transduced NK cells; seven days following their transduction, transduced NK cells expanded a median 30 fold while non transduced NK cells expanded a median 27 fold (p=n.s.). Cytotoxicity assays showed EBV-LCLs were resistant to killing by IL-2 activated T cells and in vitro expanded NK cells. In contrast, CD19CAR LV vector transduced NK cells were highly cytotoxic against EBV-LCLs; at 10:1 effector to target ratio (E:T), 43% of EBV-LCLs were killed by CD19CAR LV transduced NK cells versus 6% killing by non transduced NK cells (p=0.0002). NK cytotoxicity of K562 targets was not altered by CD19CAR LV transduction; at a 10:1 E: T ratio, LV transduced NK cells lysed 80% of K562 cells vs. 84% lysis by non transduced NK cells (p=n.s.). We next transduced IL-2 activated T-cells with the CD19CAR LV vector to compare their cytotoxicity to transduced NK cells against CD19+ LCLs. At a 10:1 E: T ratio, 11 % vs 1% of LCLs were killed by transduced vs non transduced T cells respectively (p=0.002). Although the TE of IL-2 activated T-cells was higher than NK cells (mean TE of 38 % vs 15% in T-cells and NK cells respectively, p=0.02), LV transduced NK cells were more cytotoxic to EBV-LCLs than transduced T-cells at the same E: T ratios. In conclusion, we show successful transduction of ex vivo expanded NK cells with a CD19CAR can be achieved using a LV vector, with CD19CAR transduced NK cells exhibiting enhanced antigen specific cytotoxicity. These findings provide both a method and rationale for clinical trials exploring the antitumor effects of adoptively infused CD19CAR LV transduced NK cells in patients with refractory B cell malignancies.

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Fusion Proteins of Ligands for NKG2D and CD20-Directed ScFvs Sensitize Lymphomas for NK Cell-Mediated Lysis

Blood ◽

10.1182/blood.v116.21.2843.2843 ◽

2010 ◽

Vol 116 (21) ◽

pp. 2843-2843

Author(s):

Christian Kellner ◽

Daniela Hallack ◽

Pia Glorius ◽

Matthias Staudinger ◽

Sahar Mohseni Nodehi ◽

...

Keyword(s):

T Cells ◽

B Cell ◽

Nk Cells ◽

Natural Killer ◽

Tumor Cells ◽

Mhc Class I ◽

Fusion Proteins ◽

Nk Cell ◽

Effector Cells ◽

Healthy Donors

Abstract Abstract 2843 Natural killer group 2 member D (NKG2D) is an important activating receptor controlling cytotoxicity of natural killer (NK) cells and T cells and plays an important role in immune surveillance against tumors. For redirecting NK cells to B-lymphoid tumor cells two recombinant bifunctional antibody-based fusion proteins were designed in order to coat malignant cells with ligands for NKG2D and attract NK cells. Therefore, a human CD20-directed single-chain fragment variable (scFv) was fused to NKG2D-specific ligands, either MHC class I chain-related protein A (MICA) or unique long 16-binding protein 2 (ULBP2). These two fully human fusion proteins, designated MICA:CD20 and ULBP2:CD20, respectively, were expressed in eukaryotic cells and purified to homogeneity. Size exclusion chromatography revealed that both purified proteins predominantly formed monomers. MICA:CD20 and ULBP2:CD20 specifically and simultaneously bound to CD20 and NKG2D and efficiently mediated lysis of lymphoma cell lines with mononuclear cells from healthy donors as effector cells. Analysis of the activation status of NKG2D-positive T cells and NK cells revealed that MICA:CD20 and ULBP2:CD20 activated resting NK cells, but not T cells, indicating that NK cells were the relevant effector cell population for the two molecules. In cytotoxicity assays using human NK cells from healthy donors, both agents sensitized lymphoma cell lines as well as fresh tumor cells for NK cell-mediated lysis. MICA:CD20 and ULBP2:CD20 induced lysis at low nanomolar concentrations with half maximum effective concentrations between 1 and 4 nM depending on target cells. Interestingly, ULBP2:CD20 exhibited a higher cytolytic potential than MICA:CD20 in terms of maximum lysis. Importantly, MICA:CD20 and ULBP2:CD20 induced lysis of 13/13 tested primary tumor cell samples from patients with different B cell malignancies including chronic lymphocytic leukemia, mantle cell lymphoma and marginal zone lymphoma. Interestingly, cell surface expression of endogenous MICA and ULBP2 was low or not detectable on fresh tumor cells. In addition, ULBP2:CD20 was also capable of inducing lysis of tumor cells in cytotoxicity experiments using autologous patient-derived NK cells as effector cells, indicating that the triggering signal was sufficient to overcome inhibition by interactions between killer cell immunoglobulin-like receptors and MHC class I molecules. Moreover, both MICA:CD20 and ULBP2:CD20 synergistically enhanced antibody-dependent cellular cytotoxicity (ADCC) by the monoclonal antibody daratumumab directed against CD38 which is co-expressed together with CD20 on certain B cell lymphomas. This approach of simultaneously triggering ADCC and natural cytotoxicity by these bifunctional fusion proteins may represent a promising strategy to achieve stronger NK cell-mediated antitumor responses. Disclosures: de Weers: Genmab : Employment. van De Winkel:Genmab: Employment. Parren:Genmab: Employment.

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Negative Impact Of KIR/HLA Interactions On NK Cell Activity Can Be Overcome By The Glycoengineered Antibody GA101 (Obinutuzumab)

Blood ◽

10.1182/blood.v122.21.376.376 ◽

2013 ◽

Vol 122 (21) ◽

pp. 376-376

Author(s):

Grzegorz Terszowski ◽

Christian Klein ◽

Jakob Passweg ◽

Martin Stern

Keyword(s):

B Cell ◽

Nk Cells ◽

Target Cell ◽

Cell Activation ◽

Nk Cell ◽

Negative Impact ◽

Target Cells ◽

Cd107a Expression ◽

Cd20 Antibody ◽

Anti Cd20

Abstract Antibody dependent cellular cytotoxicity (ADCC) is one of the mechanisms by which therapeutic antibodies mediate tumor cell killing. The anti-CD20 antibody rituximab is the current standard of care in the treatment of B-cell lymphomas. GA101, a novel anti-CD20 antibody, contains a glycoengineered Fc-portion allowing approximately 10-fold greater affinity to FcgR3A, the Fc-IgG receptor expressed on the majority of natural killer (NK) cells. NK cell function is also regulated by inhibitory killer-cell immunoglobulin-like receptors (KIR), which interact with HLA class I antigens (2DL1-HLA-C2; 2DL2/3-HLA-C1, 3DL1-HLA-Bw4). The KIR/HLA interaction during NK cell development leads to the acquisition of full effector function in the “licensing” process, but also provides one of the main mechanisms of NK cell tolerance. The present study analyzed how KIR/HLA interactions influence ADCC, and whether there are differences between conventional and glycoengineered antibodies. We analyzed the activation (in terms of the degranulation measured by the CD107a expression) and killing capacity of KIR-positive NK cells induced by rituximab, GA101, and the parental non-Fc modified (wild-type) GA101wt. Target cells included HLA-negative B-cell lymphoma lines or B-cell lines expressing one or more HLA molecules. We confirmed previous observations that the licensing status affects the potential for rituximab-induced ADCC (degranulation against HLA-deficient 721.221 in licensed cells 35 ± 4% versus 19 ± 3% of unlicensed cells, p<0.01); and that KIR/HLA interactions strongly and selectively inhibit the response to targets expressing cognate HLA ligands (e.g. CD107a expression in KIR3DL1+ NK cells 17 ± 3% against 721.221-Bw4 cells, compared to 32 ± 4% against 721.221, p<0.01). Next, we analyzed rituximab-induced NK cell activation in donors expressing one, two, or three KIR ligands after co-incubation with target B-cell lines expressing corresponding HLA molecules. These experiments showed that the inhibitory effect during target cell encounter dominates over the activating effect of NK cell licensing, which leads to unlicensed NK cells being the strongest effectors of ADCC with rituximab (Figure, Panel A). We next compared the effect of the KIR/HLA interaction on rituximab-, GA101wt- and GA101-induced ADCC. GA101 largely compensated the hyporesponsiveness of unlicensed cells and NK cell activation was independent of the presence of HLA KIR ligands on target cells (Figure, Panel A). Finally, we addressed the question of how levels of NK cell degranulation correspond to target cell elimination. Correlation between CD107a expression and target cell elimination was excellent for all antibodies (Figure Panel B). GA101 induced the highest level of activation and the most effective target elimination. In contrast to rituximab and GA101wt, no negative impact of KIR/HLA interaction on degranulation or target cell elimination could be detected for GA101. In summary, we show that KIR/HLA interactions are relevant for ADCC with rituximab, with the negative impact during target cell encounter dominating over the positive effect of licensing. In contrast, the novel glycoengineered GA101 antibody overrides the negative signals derived from the KIR/HLA interaction and activates all NK cell subsets. These data suggest that the Fc-modification to enhance ADCC can be an effective strategy to augment the efficacy of therapeutic monoclonal antibodies by recruiting NK cells irrespective of their inhibitory KIR expression. Disclosures: Terszowski: Roche: Research Funding. Klein:Roche Glycart AG: Employment. Stern:Roche: Research Funding.

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127 Preclinical evaluation of NKX019, a CD19-targeting CAR NK Cell

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2020-sitc2020.0127 ◽

2020 ◽

Vol 8 (Suppl 3) ◽

pp. A140-A140

Author(s):

Nadege Morisot ◽

Sarah Wadsworth ◽

Tina Davis ◽

Nicole Dailey ◽

Kyle Hansen ◽

...

Keyword(s):

T Cells ◽

B Cell ◽

Nk Cells ◽

Half Life ◽

Nk Cell ◽

Cytolytic Activity ◽

Nsg Mice ◽

Animal Care

BackgroundNatural killer (NK) cells are highly effective and fast-acting cytolytic cells capable of eradicating target cells with limited adverse effects such as cytokine release syndrome (CRS) or graft-versus-host disease. Chimeric antigen receptors (CARs)-engineered NK cells have been recently used against leukemia with encouraging clinical outcomes.1 The surface antigen CD19, expressed by B-lymphoblasts, represents an ideal CAR target against B cell acute lymphoblastic leukemia (B-ALL). We developed a highly potent CD19 -directed CAR NK cell therapy, NKX019, with an extended in vivo half-life aimed at killing CD19-expressing target.MethodsNK cells isolated from healthy PBMCs were expanded in the presence of NKSTIM cells, IL-2, IL-12, IL-18 and transduced with both a CD19-targeted CAR construct and a membrane-bound form of IL-15 (mbIL-15). Control (non-engineered) NK cells were produced in parallel. Cytotoxic activity of NKX019 against CD19+ B-ALL cell line (REH), pre-B ALL cell line (Nalm-6), allogeneic PBMCs was assessed using Incucyte® or flow cytometry. NSG mice bearing either Nalm-6.fluc (Nalm6) or REH.fluc (REH) tumor received different concentrations of NKX019 or control NK cells. In-life analysis of tumor-bearing and naïve NSG mice include: 1) bioluminescence imaging, 2) clinical observations, 3) serum cytokines and 4) CAR+ NK cell persistency.ResultsNKX019 showed enhanced cytolytic activity against REH and Nalm-6 tumor cells compared to control NK cells and CAR19+ T cells. The superiority of NKX019 over CAR19+ T cells was more pronounced at the earlier time point (24 hours) with near identical calculated EC50 observed at 72 hours for both cell types. Increased cytolytic activity of NKX019 was limited to CD19+ cells in bulk PBMCs. Consistent with our in vitro observations, NKX019 controlled Nalm-6 and REH tumor growth in doses as low as 2 × 106 cells/kg for up to 30 days with no apparent increase in cytokines commonly associated with CRS. Increased Nalm-6 tumor growth coincided with an apparent decrease in measurable NKX019 in the periphery. In tumor-naïve NSG mice, NKX019 was detectable in the blood for up to 9 weeks post-infusion consistent with its extended half-life.ConclusionsNKX019 expresses mbIL-15 and is produced in the presence of IL-12 and IL-18, resulting in enhanced in vitro expansion and longer in vivo half-life than non-engineered NK cells. NKX019 also exhibited advantages compared to CAR19+ T cells including faster cytotoxic kinetics and limited production of cytokines associated with CRS. A first-in-human trial of NKX019 in B cell malignancies is planned for 2021.Ethics ApprovalThe animal procedures described in this abstract were conducted in accordance with Explora BioLabs Animal Care and Use Protocol approved by Explora BioLabs Institutional Animal Care and Use Committee.ReferenceLiu, et al. 2020 NEJM

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A High Dose of Naïve CD8+ T-Cells Increases the Incidence of Graft-Versus-Host Disease (GvHD) after Donor Lymphocyte Infusions (DLIs) from HLA Identical Sibling Donors

Blood ◽

10.1182/blood-2019-124358 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 3292-3292

Author(s):

Guillermo Ortí ◽

Carlos Palacio ◽

Irene García-Cadenas ◽

Isabel Sánchez-Ortega ◽

María-José Jimenez ◽

...

Keyword(s):

T Cells ◽

B Cell ◽

Nk Cells ◽

Board Of Directors ◽

Research Funding ◽

Nk Cell ◽

Mixed Chimerism ◽

High Dose ◽

Cellular Composition ◽

Advisory Committees

Introduction DLIs represent a major therapeutic approach for relapse and mixed chimerism (MC) after allogeneic hematopoietic cell transplant (AlloHCT). DLI studies have identified several variables with impact on response and GvHD. Despite some studies having explored the role of T-cells and other cell subsets, such as mononuclear cells (MNCs), comprehensive data regarding the cellular composition of DLI and its role in GvHD remains incomplete, as the development of GvHD post DLI is often unpredictable. Herein we analyzed the cellular composition of DLI from fully human leukocyte antigen (HLA) identical sibling (HLA Id Sib) donors and its impact on the development of GvHD in patients who underwent AlloHCT for hematological malignancy, and its impact on the development of GvHD. Methods Inclusion criteria were as follows: 1) Patients ≥ 18 years-old, 2) AlloHCT, 3) HLA Id Sib donor; 4) treatment with DLI; and 5) signed informed consent of patient and donor. Exclusion criteria were: 1) unrelated or mismatched related donors, 2) HCT2 prior to DLI, or 3) GvHD at DLI. For the purpose of avoiding bias, only the cell composition of the first DLI (DLI1) was analyzed. The following cell subsets of the DLI were studied: CD3+, CD4+, CD8+, CD16+CD56+CD3+ (NKT-cell), CD3+CD45RA+CCR7+ (TN), CD3+CD45RA+CCR7+CD31+ (TRTE), CD3+CD45RA+CD95+CD27+ (TSCM), CD3+CD45RA-CCR7+ (TCM), CD3+CD45RA-CCR7- (TEM), CD3+CD45RA+CCR7- (TTE), CD3+CD4+CD25brightCD127dim (TREG), CD3+CD4+CD25brightCD45RA+CD127dim (naïve TREG). The TN, TCM, TEM and TEM compartment was analyzed for both CD4+ and CD8+. We also analyzed the MNCs, CD19+ (B-cell), CD27+CD19+ (mature B-cell), CD16+CD56+CD27- (natural killer (NK+) cell) and CD16+CD56+CD27+ (CD27+NK+cell). Results Fifty-six DLIs were infused in 36 patients; the median number of DLI was 1 per patient (range, 1-3). Diagnoses were as follows: 13 AML/MDS, 6 HL, 5 MPN, 4 NHL, 4 CLL, 3 MM and 1 B-ALL. For the study, a landmark analysis was performed from the DLI date. The median follow up from DLI was 282 days (range, 9-5,560 days). Overall response rate in relapsed patients was 29% (9 of 31 patients; 6 CR and 3 PR, most responses being observed after DLI1. Further, five patients had DLI for MC and full donor chimerism was achieved in all patients. Thirteen patients (36%) developed GvHD post DLI. Two patients had GvHD before DLI, but there was no case of GvHD at DLI. The median time interval form DLI to GvHD was 76 days (range, 7-261). As per clinical presentation, 10 patients (27%) had acute GvHD, whereas eight patients (22%) had chronic GvHD. The 6-month and 1-year cumulative incidence (CI) of GvHD was 33% and 46%, respectively. When the risk of GvHD was analyzed according to DLI cell subsets, we observed that a DLI1 containing >3x106 CD8+TN correlated with an increased incidence of GvHD (Figure 1a). Also, a DLI1 with >0.8x108 MNCs/Kg (Figure 1b), >2.6x106 mature B-cell/Kg, or >0.35x106 CD27+NK+cells/Kg were linked to the development of GvHD (Table 1). Noteworthy, CD3+, TN (both CD4+ and CD8+ combined) or CD4+TN had no impact on the development of GvHD; and a high proportion of TREG was not protective for the development of GvHD (Table 2). Finally, there was no statistically significant association between any clinical variable and GvHD. Conclusion In conclusion, in this cohort of AlloHCT patients from HLA Id Sib donors, a DLI1 containing a high proportion of CD8+TN, but not CD4+TN, increased the probability of developing GvHD. Further, a DLI1 containing a high dose of MNCs, CD27+NK+cells and mature B-cell also associated with GvHD. These data provide novel insight for the understanding of GvHD post DLI. A DLI1 containing a lower dose of CD8+TN could reduce the risk of GvHD, but this asset warrants further validation in larger cohorts, and within a controlled randomized trial setting. Disclosures Bosch: F. Hoffmann-La Roche Ltd/Genentech, Inc.: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Kyte: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Celgene: Honoraria, Research Funding; Acerta: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; AbbVie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Takeda: Honoraria, Research Funding; AstraZeneca: Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau.

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Influence of Different Components of the Tumor Microenvironment on Human Patient-Derived Lymphoma Cell Engraftment in Immmunodeficient Mice

Blood ◽

10.1182/blood.v126.23.1459.1459 ◽

2015 ◽

Vol 126 (23) ◽

pp. 1459-1459

Author(s):

Cordula Tschuch ◽

Kerstin Klingner ◽

Dorothee Lenhard ◽

Cyrill Rentsch ◽

Christian Wetterauer ◽

...

Keyword(s):

T Cells ◽

B Cells ◽

Tumor Growth ◽

Nk Cells ◽

Nk Cell ◽

Subcutaneous Implantation ◽

Lymphoma Cells ◽

Cell Engraftment ◽

Nog Mice

Abstract Patient-derived xenografts (PDX) have become reliable tools for preclinical research for many types of cancer. We recently reported the establishment of 4 human diffuse-large B-cell lymphoma (DLBCL) PDX in mice arising from patient prostate carcinoma tissue (Wetterauer,C. et al. 2015). All PDX were EBV associated and of ABC subtype as determined by the new algorithm of Choi et al. 2009. In contrast, none of the donor patients showed clinical or histological evidence of lymphoma disease. To address the question why lymphoma engraftment takes place in immunodeficient mice whereas patients do not show clinical evidence of disease, we compared growth behavior of the DLBCL PDX in 4 different mouse strains harboring different immunological deficiencies. Furthermore using 3 different implantation routes the dissemination behavior of these models into different immunological niches was evaluated. Results were compared to a similar study carried out with an EBV negative DLBCL PDX of a secondary, cerebral lymphoma (ABC-subtype). None of 4 EBV+ DLBCL PDX showed tumor growth after implantation in NMRI nu/nu (B- cells+, T cells-, NK cells+, n=5 mice/PDX) or 1147-F mice (B cells-, T cells+, NK cells+ n=5 mice/PDX). Subcutaneous implantation into NOD/SCID mice (B cells-, T cells-, NK cells+ n=5 mice/PDX) revealed tumor growth for 2 of 4 lymphomas displaying take rates of 80% (4 out of 5 mice) and 100% (5 out of 5 mice) within one model. Infiltration of murine NK cells in tumor tissue was determined by IHC (analyses ongoing). All 4 EBV+ lymphoma models showed stable growth in NOG mice (B cells-, T cells-, NK cells- n= 10 - 12 mice/PDX) with a take rate of 100% and an average passaging time of 20 days, ranging from 14 to 26 depending on the specific model. Taken together these results indicate that tumor growth of EBV+ lymphomas in mice depends on the NK cell activity as well as the B- and T cells status of the recipient mouse. These cell populations control tumor engraftment in mice, which might explain the absence of lymphoma disease in donor patients. In contrast the PDX of a clinically apparent EBV- DLBCL patient was able to circumvent NK cell block in NMRI nu/nu mice and showed similar take rates (100%, 5 out of 5 engrafted mice) but prolonged passaging times (35 days) as compared to NOG (26 days). Comparison of tumor cell engraftment after subcutaneous (s.c.), intravenous (i.v.) and intratibial (i.t.) injection of tumor cells in NOG mice revealed disseminated tumor growth for the EBV- DLBCL PDX exclusively when injected i.v. or i.t.. After subcutaneous implantation of lymphoma material, no tumor cells could be detected by flow cytometry or IHC in lymph nodes, blood, spleen, brain, liver, lung or bone marrow of recipient mice. However, lymphoma cells could be detected in liver and bone marrow of all (n= 8) i.v. and i.t. injected mice. Currently we are analyzing the dissemination behavior of the EBV+ lymphomas after injection into the different immunological niches as described above. In summary these data highlight the importance of the tumor microenvironment in lymphoma engraftment and dissemination in vivo. Accordingly we could show for our EBV associated lymphoma models that NK-, T- and B cell population control engraftment in vivo and are possibly involved in controlling EBV+ transformed lymphatic cells in patients. Furthermore, we showed that injection into the biologically relevant niche enables lymphoma cells to form multiple tumor nodules in distinct organs in vivo. These models contribute to a better understanding of the interplay between lymphoma cells and their microenvironment. They will thereby facilitate the discovery of new targets for innovative anti-lymphoma treatment strategies. Disclosures Tschuch: Oncotest GmbH: Employment. Klingner:Oncotest GmbH: Employment. Lenhard:Oncotest GmbH: Employment. Haapaniemi:Biositehisto Oy: Employment. Schueler:Oncotest GmbH: Employment.

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Mechanisms of Antigen Dependent and Independent Rejection of High Grade B-Cell Lymphoma in a Murine Model.

Blood ◽

10.1182/blood.v114.22.4091.4091 ◽

2009 ◽

Vol 114 (22) ◽

pp. 4091-4091

Author(s):

Armin Gerbitz ◽

Madhusudhanan Sukumar ◽

Florian Helm ◽

Andrea Wilke ◽

Thomas Kammertoens ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

B Cell ◽

Nk Cells ◽

Mhc Class I ◽

Cell Lines ◽

Interferon Gamma ◽

Class I ◽

High Grade ◽

Lymphoma Cells

Abstract Abstract 4091 Poster Board III-1026 The rejection of Non Hodgkin Lymphomas expressing foreign, for example viral, antigens is compromised despite the presence of specific T-cells. The underlying immunosuppressive mechanisms are poorly understood. Using a transgenic mouse lymphoma model, where the proto-oncogene c-myc is driven by parts of the immunoglobulin lambda locus representing a t(8;22) translocation as found in Burkitt's lymphoma, we investigated the anti-lymphoma activity of specific T-cells. By retroviral transduction of a specific foreign antigen (chicken Ovalbumin-IRES-GFP, OVA) into cell lines from primary c-myc transgenic lymphomas we established a syngeneic model that would allow us to address the role of interferon gamma signalling in rejection of high grade lymphomas. All primary lymphoma cells displayed normal MHC class I and II levels on the surface when compared to wildtype splenic B-cells. This expression could be enhanced by treatment with interferon gamma (IFNg,100U/ml) up to 10 fold. When retrovirally OVA-transduced lymphoma cells were injected into either wildtype or GFP transgenic recipients, animals displayed a significant delay in lymphoma growth compared to non transduced or IRES-GFP transduced control cell lines. 80% of the recipient mice rejected OVA expressing lymphomas. By contrast, we observed 100% mortality when GFP expressing control lymphomas were injected in GFP transgenic recipients, which are tolerant for GFP. Developing OVA expressing lymphomas (20%) displayed a loss of GFP expression indicating a selection for antigen negative cells (p=0.001). In spleens from mice rejecting OVA-expressing lymphomas we found up to 1.8% SIINFEKL specific T-cells. To gain more mechanistic insights, we transferred OVA expressing lymphoma cells into IFNg, Stat1-/- or IFNg-/-receptor deficient recipients. Lack of STAT1-/- or IFNg-receptor on the recipient side or inability to secrete IFNg was associated with fast lymphoma progression and growth was not different when compared to non transduced, antigen negative cell lines. When IFNg-receptor or STAT1 deficient OVA expressing cell lines were transferred into wildytpe mice, rejection was not influenced. Outgrowing OVA expressing lymphomas in wildtype mice displayed a high MHC class I and II expression compared to the cell line prior to injection. MHC induction was absent in lymphomas transferred to Stat1 or IFNg deficient recipients. Depletion of NK cells by anti AsialoGM antibody in wildtype recipients resulted in a significant reduction of disease free survival (80% vs. 50%, p=0.002) and animals developed larger tumors which were eventually rejected resulting in a comparable overall survival. In peripheral blood of NK depleted mice significantly more OVA specific T-cells were detectable through pentamer staining. When lymphoma cell lines were injected into Rag1-/- mice, NK cell mediated rejection was also significantly impaired upon depletion. Our results suggest that T-cell mediated rejection of high grade B-cell lymphomas is strongly dependent on host IFNg secretion and that NK cells substantially contribute to T-cell mediated rejection. Disclosures: No relevant conflicts of interest to declare.

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Siglec-7 Tetramers Characterize B Cell Subpopulations and Identify Immunomodulatory Ligands on Malignant Cells

Blood ◽

10.1182/blood.v116.21.1728.1728 ◽

2010 ◽

Vol 116 (21) ◽

pp. 1728-1728

Author(s):

Philippa Mang ◽

Friederike Gieseke ◽

Susanne Viebahn ◽

Inga Gondesen ◽

Anne Kruchen ◽

...

Keyword(s):

T Cells ◽

Cell Surface ◽

B Cell ◽

Nk Cells ◽

Immune Evasion ◽

Nk Cell ◽

Sialic Acids ◽

Target Cells ◽

Malignant Cells ◽

Neuraminidase Treatment

Abstract Abstract 1728 Cell surface glycoconjugates have important functions in the modulation of immune cell maturation, activation and homeostasis. Glycans on cell surfaces are generally sialylated. Sialic acids are predominantly found on the non-reducing end of oligosaccharide chains of glycoconjugates. They are recognized by sialic acid-binding immunoglobulin-like lectins (siglecs). CD33-related siglecs are receptors predominantly expressed on immune cells. Most of the CD33-related siglecs display a tyrosine-based inhibitory motif (ITIM) known to downregulate effector cell functions. So far, identification of ligands for siglecs was hampered by low affinity between proteins and glycans, but by glycan array analysis α2,8-, α2,3- as well as α2,6-linked sialic acids were identified as the carbohydrate part of the ligand. In order to analyse the expression of physiological siglec ligands on different lymphocyte subpopulations, the extracellular three domains of siglec-7 were cloned. To allow for mammalian glycosylation, the protein was expressed in 293T cells. To overcome the low affinity of lectins, siglec-7 was enzymatically biotinylated and oligomerized using streptavidin in analogy to HLA tetramers. Flow cytometric analysis of lymphocytes revealed reproducible binding patterns of siglec-7 tetramers. Specificity was ensured by treatment of PBMC with neuraminidase, specifically cleaving terminal sialic acids in α2,3, α2,6 and α2,8 linkages. On T cells, the density of siglec-7 ligands increased 3.5-fold during activation. This is contradictory to results using plant lectins, like Maackia amurensis lectin and Sambucus nigra lectin, which indicated an 1.8- and 1.5-fold decrease, respectively. Activation of NK cells did not affect the siglec-7 ligand expression. Interestingly, B cells could be divided in a siglec-7 ligand positive and a siglec-7 ligand negative population. This may reflect different differentiation or activation stages, which were not found with any antibody combination directed against typical protein B cell markers. Malignant transformation is often associated with aberrant cell surface glycosylation. In acute lymphoblastic leukemia (ALL), blasts of the more aggressive T-ALL express 17 fold higher amounts of siglec-7 ligand than blasts of the B cell lineage (cALL), where the siglec-7 ligand was nearly not detectable. These findings may contribute to the poor prognosis of T-ALL by a possible immune escape achieved by siglec-ligand binding induced inhibition. Since immune evasion mechanisms are also described for other malignancies, we additionally analysed rhabdomyosarcoma (RMS) cells. Siglec-7 ligand could be found on all tested RMS cell lines in different expression levels, independent of the classification. Siglec-7 is a known inhibitory receptor expressed by NK cells and T cells. The effect of siglec-7 ligand expressed by malignant cells on NK cell cytotoxicity was analysed. By the removal of all terminal sialic acids on the surface of RMS cells by neuraminidase treatment, the NK cell-induced cytotoxicity could be increased by 20%. Coating of siglec-7 ligands on target cells by preincubation with recombinant siglec-7 protein also resulted in an increased cytotoxicity. These results show that sialic acids play an important role in the immune system as physiologic ligands. However, the expression of siglec-7 ligands on malignant cells may contribute to immune evasion mechanisms. Disclosures: No relevant conflicts of interest to declare.

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