EX VIVO NK CELL DIFFERENTIATION FROM CD34+ HEMATOPOIETIC CELLS

Abstract

The present invention relates to the ex vivo differentiation of NK cells from CD34+ hematopoietic stem cells. Such NK cells and their progenitor cells can be used in therapies of a broad range of malignancies. In the present invention it is shown that IL-12 modulates ex vivo NK cell differentiation. Specific, we achieved significantly higher expression of KIR, CD16 and CD62L in the presence of IL-12 in the cell culture system. The induction of receptor expression by IL-12 occurred predominantly on an augmented population of CD33+NKG2A+ NK cells early during NK cell differentiation. These cells further show enhanced cytolytic activity against MHC class I positive AML targets. In line with the enhanced CD16 expression, IL-12 modulated ex vivo generated NK cells exhibit an improved antibody-dependent-cytotoxicity, using anti CD20 antibody on various B cell targets. Additional to the enhanced expression of CD62L, we show that this cell population consists of a specific chemokine receptor profile. By showing an increased capacity for adhesion to lymphendothelial cells and a specific chemokine receptor profile, we show that IL-12 provided the ex vivo generated NK cells with specific tissue-homing abilities.

Claims

1. A method for producing NK cells said method comprising iproviding a sample of human CD34 positive cells, iiexpanding said CD34 positive cells ex vivo, iiiculturing CD34 positive cells obtained in step ii ex vivo in an NK-cell differentiation medium, said method characterized in that said NK-differentiation medium comprises IL-12.

2. A method according to claim 1, wherein said NK-differentiation medium comprises between 20 pgram/ml and 20 ngram/ml IL-12.

3. A method according to claim 2, wherein said NK-differentiation medium comprises between 0.2 ngram/ml and 2 ngram/ml IL-12.

4. A method according to claim 1, wherein step ii) is performed with a culture medium comprising three or more of stem cell factor (SCF), flt-3Ligand (FLT-3L), thrombopoietin (TPO) and interleukin-7 (IL-7) and three or more of granulocyte-macrophage-colony-stimulating factor (GM-CSF), granulocyte-colony-stimulating factor (G-CSF), interleukin-6 (IL-6), leukaemia-inhibitory factor (LIF) and Macrophage-inflammatory protein-1 alpha (MIP-I alpha).

5. A method according to claim 1, wherein step iii) is performed with an NK-cell differentiation medium comprising one or more of IL-2 and IL-15; and one or more of IL-7 and SCF; and three or more GM-CSF, G-CSF, IL-6, LIF and MIP-I alpha.

6. A method according to claim 1, wherein step ii) is performed in culture medium comprising low molecular weight heparin.

7. A collection of NK-cells obtainable by a method according to claim 1.

8. A collection of NK-cells according to claim 7, wherein at least 20% of the CD56 positive cells express CD62L; at least 10% of the CD56 positive cells express KIR; or at least 20% of the CD56 positive cells express CD16.

9. A collection of NK-cells according to claim 8, that comprises NKG2A and CD33 positive cells and wherein at least 50% of the CD56 positive, NKG2A positive and CD33 positive cells are positive for both KIR and CD62L.

10. A cell bank comprising a collection of NK-cells according to claim 7.

11. A method for killing cancer cells with NK-cells, said method characterized in that said NK-cells comprise a collection according to claim 7.

12. A collection of NK-cells according to claim 7, for the preparation of a cell transplant.

13. A collection of NK-cells according to claim 7, for use in the treatment of cancer, wherein preferably said cancer treatment further comprises treatment with an antibody specific for an antigen present on cells of said cancer.

14. A collection of NK-cells according to claim 7, for use in the treatment of cancer, wherein said cancer treatment further comprises treatment with an antibody specific for an antigen present on cells of said cancer, and wherein said cancer is a cancer of hematopoietic origin.

15. A collection of NK-cells according to claim 7, for use, in the treatment of cancer, wherein preferably said cancer treatment further comprises treatment with an antibody specific for an antigen present on cells of said cancer, and wherein said antibody is specific for CD20.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0040] FIG. 1. Scheme of established and modulated ex vivo NK cell differentiation protocol.

[0041] The previously established as well as the IL-12 modulated ex vivo hematopoietic stem cell (HSC) expansion and NK cell differentiation method is shown. In the basic protocol CD34.sup.+ UCB cells were expanded by SCF, IL-7, TPO, Flt3L, G-CSF, GM-CSF, IL-6 and low molecular weight heparin for 10 days, followed by the differentiation of CD56.sup.+ NK cells by replacement of TPO with IL-15 at day 10 and Flt3L and the low molecular weight heparin by IL-2 at day 14. For the modulation of the culture system with IL-12, at day 10 of culture NK cell differentiation was induced by IL-15 alone (a) or by IL-15 and IL-12 (b). Cells were grown up to a total of at least 28 days.

[0042] FIG. 2. Effects of IL-12 on the phenotype and the purity of ex vivo generated NK cells.

[0043] Effects of high and low dose IL-12 on the ex vivo NK cell generation culture and the NK cell phenotype were analyzed by flow cytometry and cell counting. In a titration analysis the effect of low (10 pg/ml) to high (20 ng/ml) concentrations of IL-12 on NK cell purity (A) and NK cell receptor expression (B) were determined by cell counting and flow cytometry analysis for CD56 expression and NKG2A CD62L, CD16 and KIR expression on CD56.sup.+ cells.

[0044] Values are shown as meanSD calculated from triplicate wells for one representative experiment at day 22 of culture. (C) A concentration of 0.2 ng/ml IL-12 was chosen for further experiments and analyzed at day 29 of culture for reduction in total cultured cells and CD56.sup.+ NK cell purity by cell counting and flow cytometry for CD56.sup.+ cells. Mean percentage SEM for several independent cultures (n) are shown as indicated.

[0045] FIG. 3. Comparison of receptor expression correlated with cytotoxicity and homing on ex vivo with and without IL-12 differentiated NK cells.

[0046] The effect of 0.2 ng/ml IL-12 on the expression of several NK cell antigens was determined by flow cytometry analysis at day 29 of ex vivo differentiation. Flow cytometry dot plots depicting the expression of CD62L, KIR, CD16 and NKG2A on gated CD56.sup.+ cells (A) as well as for CCR1, CCR6-8 and CD56 (B) are shown for one representative ex vivo NK cell differentiation culture induced with and without IL-12. The statistical comparison, determined by flow cytometry analysis for 5 independently performed experiments of ex vivo NK cell differentiation generated with or without 0.2 ng/ml IL-12, is displayed for CD62L, KIR and CD16 as mean percentage SEM (C).

[0047] FIG. 4. Effect of IL-12 on distinct CD33 and NKG2A determined stages of NK cell differentiation.

[0048] Ex vivo with or without 0.2 ng/ml IL-12 differentiated NK cells were analyzed for their CD33 and NKG2A maturation profile at day 28 of culture. Comparative flow cytometry dot plots revealing the expression of CD33 and NKG2A are shown on the left upper panels indicating the gates for CD33.sup.+/NKG2A, CD33.sup.+/NKG2A.sup.+, CD337NKG2A.sup.+ and CD337NKG2A cells that were further analyzed for KIR and CD62L expression in the consecutive boxed panels. A representative example of 3 cultures analyzed is shown.

[0049] FIG. 5. CD56 expression profile of ex vivo differentiated IL-12 modulated NK cells.

[0050] The expression level of CD56 in correlation with CD62L, NKG2A, KIR and CD16 was compared in flow cytometry analysis for gated CD56.sup.+ ex vivo differentiated NK cells with or without IL-12 modulation during culture. One representative experiment is shown that revealed particular strong induction levels for CD62L, KIR and CD 16.

[0051] FIG. 6. Analysis of adhesive and migratory capacities of ex vivo differentiated and IL-12 induced NK cells.

[0052] (A) Comparison of ex vivo generated NK cells, that were generated with or without 0.2 ng/ml IL-12, for adhesion to lymphatic endothelial cells. Ex vivo generated NK cells from day 28 of culture were purified and subsequently used in adhesion assays on lymphatic endothelial cells (LecTERT) or human umbilical vein endothelial cells (HUVEC). Mean percentage values SEM calculated from 3 independent experiments each performed in duplicate are shown.

[0053] FIG. 7. Cytotoxic capacities of ex vivo differentiated NK cells in correlation with IL-12 modulation during culture.

[0054] NK cells stimulated with or without IL-12 were cultured with K562 (light grey bars) or KG1a (dark grey bars) at an effector: target ratio (E:T ratio) from 1:1 overnight for 18 h. Co-cultures were analyzed for cytotoxicity (A) or CD107a degranulation (B).

[0055] FIG. 8. Relation of enhanced antibody-dependent-cytotoxicity and IL-12 modulation during ex vivo NK cell generation.

[0056] Ex-vivo generated NK cells from day 28 of culture with and without induction of 0.2 ng/ml IL-12 were purified and subsequently used in Europium-release killing assays. B-cell target cell lines 7221.221, REH, UoCB6, Nalm-6 and SEM were used at several effector to target ratios and previously labeled with the therapeutically used antibody Rituximab if indicated. Mean values SD calculated from triplicate wells are shown for a representative experiment performed.

[0057] FIG. 9: UCB-NK cells in combination with rhIL-15 mediate antileukemic response in vivo.

[0058] Adult NSG mice were injected in their right femur with 10.sup.5 Luciferase-expressing K562 AML cells. The day after, mice were treated with 2010.sup.6 UCB IL-12-NK cells i.v. combined with IL-15 i.p. administration (0.5 microgram/mouse i.p. every 2-3 days for 14 days), or received PBS or IL-15 alone as control (n=6 per group). Tumor load was monitored by bioluminescence imaging from day 8 after AML cell inoculation and next every 3-4 days. (A) BLI at day 15 after tumor cell injection. (B) In vivo tumor load follow-up by BLI, meanSD (C) Time to first tumor detection (D) Survival curve.

EXAMPLES

Materials and Methods

CBMC Isolation and Enrichment of CD34.SUP.+ Stem and Progenitor Cells

[0059] Human umbilical cord blood (UCB) samples have been obtained at birth after normal full-term delivery and written informed consent with regard of scientific use and were supplied by VivoCell Biosolutions AG (Graz, Austria) within AKH Wien, Austria or from the cord blood bank of the Radboud University Nijmegen Medical Center (RUNMC, Nijmegen, The Netherlands). Mononuclear cells were isolated by density gradient centrifugation (LSM 1077 Lymphocyte Separation Medium, PAA Laboratories GmbH, Graz, Austria) and labeled with CliniMACS CD34 reagent (Miltenyi Biotech, Bergisch-Gladbach, Germany). The CD34.sup.+ cell selection was performed according the manufactures instructions and after the enrichment procedure, the CD34.sup.+ cell fraction was collected, and the cell number and purity were analyzed by flow cytometry. Finally, the obtained CD34.sup.+ UCB cells were used directly for the NK cell generation bioprocess.

Ex Vivo Expansion and Differentiation of CD34.SUP.+ Progenitor Cells

[0060] CD34.sup.+ UCB cells were transferred into culture plates and expanded and differentiated according to culture method III as described previouslyl.sup.5. In short, UCB cells were labeled with CliniMACS CD34 reagent (Miltenyi Biotech, Bergisch-Gladbach, Germany) and CD34.sup.+ cells were selected by magnetic isolation (Miltenyi MACS Separator) according instructions of the manufacturer. CD34.sup.+ cells were collected, cell number and purity established by flow cytometry and the cells used for NK cell generation. CD34.sup.+ UCB cells were transferred into culture plates and expanded and differentiated according to culture method III as described previously.sup.15. In short, CD34.sup.+ cells were expanded for 10 days in GB GM supplemented with a high dose of the factors SCF (27 ng/ml, CellGenix, Freiburg, Germany), IL-7 (25 ng/ml, Stemcell Technologies, Grenoble, France), TPO (25 ng/ml, Stemcell Technologies), Flt3L (25 ng/ml, CellGenix) and a low dose of the factors G-CSF (250 pg/ml, Stemcell Technologies), GM-CSF (10 pg/ml, Stemcell Technologies) and IL-6 (50 pg/ml, CellGenix) as displayed in FIG. 1A. Differentiation was induced by replacing TPO by IL-15 (20 ng/ml, CellGenix) at day 10 and Flt3L by IL-2 (1000 U/ml, Chiron, Miinchen, Germany). During the first 14 days of culture low molecular weight heparin (25 mg/ml, Abbott, Wiesbaden, Germany) was included in the growth medium. Cells were grown up to a total of at least 28 days. For induction, rh-IL12 (Immunotools, Friesoythe, Germany) was added from day 10 on at a concentration of 0.2 ng/ml (if not indicated differently). For functional studies the ex vivo generated NK cells were purified with CD56 microbeads (Miltenyi Biotec) according the manufactures instructions and directly used in functional assays.

Cell Lines

[0061] Cell line K562 (LGC Standards, Wesel, Germany) was cultured in Iscove's modified Dulbecco's medium (IMDM; Invitrogen, Carlsbad Calif., USA) containing 50 U/ml penicillin, 50 g/ml streptomycin and 10% fetal calf serum (FCS; Integro, Zaandam, the Netherlands). Human B cell precursor leukemia cell lines 721.221, SEM, REH, Nalm-6 and UoC-B6 were cultured in RPMI-1640 (Sigma-Aldrich, Vienna, Austria) containing 50 U/ml penicillin, 50 g/ml streptomycin (PAA Laboratories GmbH, Graz, Austria) and 10% fetal calf serum.

[0062] Lymphatic endothelial cells stably transfected with hTERT (LecTERT) were kindly provided by Prof. Dr. Dontscho Kerjaschki AKH Vienna, Austria, cultured in DMEM medium (Invitrogen, Fisher Scientific GmbH, Vienna, Austria) containing 50 U/ml penicillin, 50 g/ml streptomycin (PAA Laboratories GmbH, Graz, Austria) and 20% fetal calf serum and were selected with 100 g/ml Hygromycin (Invitrogen, Fisher Scientific GmbH, Vienna, Austria).

[0063] Human umbilical vein endothelial cells (HUVECs) were isolated as described previously.sup.28 and cultured in EGM-2 medium (Bio Whittacker, Lonza, Verviers, Belgium).

Flow Cytometry

[0064] Cell numbers and expression of cell-surface markers were determined by flow cytometry. For immunophenotypical staining, cells were after incubation with FcR-blocking reagent (Miltenyi Biotec), incubated with the appropriate concentration of antibodies for 30 min at 4 C. After washing, expression was measured using a FACSCalibur and analyzed with CellQuestPro software (both from BD Biosciences). To determine purity and phenotype of the cultured cells following antibodies were used: CD3-FITC clone UCHT1 (Immunotools), CD56-APC clone NCAM16.2 (BD Biosciences), NKG2A-PE clone Z199.1.10 (Beckman Coulter), CD16-PE clone 3G8 (BD Biosciences), CD62L-FITC clone LT-TD180 (Immunotools), KIR-FITC clone 180704 (R&D Systems), CXCR3 (R&D Systems), CXCR4 (Biolegend), CXCR5 (R&D Systems), CCR1 (R&D Systems), CCR7 (R&D Systems), CCR6 (Biolegend).

Adhesion Assay

[0065] Ex vivo generated and purified NK cells were transferred onto confluent LecTERT cells and incubated in RPMI-1640 for 30 min at RT on a belly dancer. After extensive washing, cells were trypsinized, stained with CD56 APC and analyzed as described under Flow Cytometry.

Cytotoxicity Assay

[0066] Flow cytometry-based cytotoxicity assays were performed as described previously.sup.14, 15. Briefly, after incubation for 4 h or overnight at 37 C., 50 l supernatant was collected and stored at 20 C. for later use to measure cytokine production. Cells in the remaining volume were harvested and the number of viable target cells was quantified by flow cytometry. Target cell survival was calculated as follows: % survival={[absolute no. viable CFSE.sup.+ target cells co-cultured with NK cells]/[absolute no. viable CFSE.sup.+ target cells cultured in medium]}*100%. The percentage specific lysis was calculated as follows: % lysis={100[% survival]}. Degranulation of NK cells during co-culture was measured by cell surface expression of CD 107a.sup.29. After 18 hrs of incubation at 37 C., the percentage of CD107a.sup.+ cells was determined by flow cytometry.

Antibody-Dependent-Cytotoxicity Assay Using Rituximab

[0067] The antibody-dependent cytotoxic activity against several human B cell precursor leukemia cell lines 721.221, SEM, REH, Nalm-6 and UoC-B6 was measured in triplicates within a Europium-release kilhng-assay as described previously.sup.30. Target cells were labelled with EuDTPA (europium diethylenetriaminopentaacetate), subsequently washed and incubated with 10 g/ml Rituximab (kindly provided by AKH Vienna, Austria) for 1 h at RT. After extensive washing 210.sup.3 target cells were incubated for 4 h with purified NK effector cells at various E:T ratios in RPMI-1640 without phenolred (PAA Laboratories, Pasching, Austria) supplemented with 10% FCS. Maximal EuDTPA release was determined by incubation with 1% Triton X-100. Values for specific release of EuDTPA were determined with Delfia Enhancement Solution (Perkin Elmer, Brunn am Gebirge, Austria) via time-resolved fluorescence. The specific cytotoxicity was calculated as percent specific EuDTPA release=(Mean sampleMean spontaneous release)/(Mean maximal releaseMean spontaneous release)100.

Statistics

[0068] Results from experiments performed in triplicates are described as meanstandard deviation of the mean (SD). Results from individual experiments are shown as meanstandard error of the mean (SEM). Statistical analysis was performed using Student's t-test. A p-value of <0.05 was considered as statistically significant.

Results

Low Dose IL-12 Enhance Expression of CD16, KIR and CD62L NK Cell Antigens During Ex Vivo NK Cell Differentiation

[0069] Initially, we aimed to analyze the impact of a various of cytokines like IL-12, IL-18 or IL-21 on our recently established and characterized ex vivo human NK cell differentiation method in addition to the use of IL-15 and IL-214, 15, to lead to a tailored NK cell phenotype. For the cytokines IL18 and IL-21 we have not found a significant improvement regarding expansion or activation of the ex vivo generated NK cell product (data not shown). However, we found that low doses of IL-12 could significantly modify the NK cell generation procedure. During culture, at day 10 after expansion of hematopoietic stem cells IL-15 and IL-12 were simultaneously added to induce NK cell differentiation (FIG. 1). We analyzed in detail the effect on NK cell differentiation of different IL-12 concentrations ranging from 10 pg/ml to 20 ng/ml. The percentage of NK cells within the culture system decreased with increasing concentrations of IL-12 (FIG. 2A), whereas the expression of CD62L, CD16 and KIR on CD56+ NK cells was elevated with higher doses of IL-12 (FIG. 2B). This dose-response analysis revealed that a concentration of 0.2 ng/ml IL-12 was enough to significantly enhance surface receptor expression on the ex vivo generated NK cells, but does not result in a significant lower purity of the final NK cell product (FIG. 2A). Having selected the most optimal IL-12 concentration purity and further experiments revealed, that the overall impact of 0.2 ng/ml IL-12 on the culture system reveals a tolerable reduction in total cell counts rather than an impact on the NK cell purity itself (FIG. 2C).

[0070] After we determined the optimal concentration of IL-12 we analyzed in more detail the impact of this cytokine on the phenotype of the ex vivo differentiated NK cells. On account of the potential therapeutical use of the ex vivo generated NK cells we focused our observations on receptors that are related to the cytotoxic activity of NK cells and receptors that are relevant for migration abilities of NK cells. Firstly, IL-12 enhanced the expression of the activating antibody-dependent-cytotoxicity receptor FcRyIII/CD16 and the expression levels of KIRs compared to NK cells generated with the basal culture system (FIG. 3A). Secondly, L-Selectin and a specific chemokine receptor repertoire of CCR6, CCR7, CXCR3, CXCR4 and CXCR5 exhibited high expression on IL-12 modulated ex vivo differentiated NK cells (FIG. 3B). In summary, the overall phenotype of NK cell modulated with IL-12 during ex vivo differentiation reveals a tailored generation of NK cells expressing CD62L, CD16, KIR, CCR1, CCR6, CCR7, CXCR3, CXCR4 and CXCR5.

IL-12 Forces a Faster Transition of CD33+NKG2A Towards CD33+NKG2A+ CD56+ NK Cells of Development Stages within the Ex Vivo NK Cell Differentiation Culture

[0071] NK cell are classically divided into CD56.sup.bright and CD56.sup.dim NK cells, which both exhibit specialized receptor expression and correlated functions. The influence of IL-12 on the expression of CD62L and particularly KIR and CD16 posed the question if this phenotype is correlated with a more mature stage of NK cell differentiation, since these NK cell antigens are most prominently expressed on the mature CD56.sup.dim peripheral blood NK cells. Recently, we described NK cell developmental subsets described by the expression of CD 33 and NKG2A.sup.31. When we compared the composition of development stages determined by the expression of CD33 and NKG2A we observed a higher proportion of the more mature CD33.sup.+NKG2A.sup.+ NK cells in IL-12 modulated (63%) than normal cultures (39%) but a lower percentage of CD33.sup.+NKG2A (23% vs. 46%) CD56+ NK cells (FIG. 4). Furthermore, the enhanced expression of CD62L and KIR observed within this enlarged proportion of CD33.sup.+NKG2A.sup.+ NK cells emphasizes that a higher proportion of the IL-12 induced ex vivo generated NK cells reside in an advanced developmental stage (FIG. 4, boxed panels).

[0072] In IL-12 modulated NK cell differentiation cultures exhibiting particular high induction levels of CD62L, CD16 and KIR we could also identify a correlation with the appearance of a CD56.sup.dim phenotype. Flow cytometry analysis revealed, that some cultures exhibited CD56.sup.dim NK cells accountable for the enhanced CD62L, CD16 and KIR expression by the ex vivo generated CD56.sup.+ NK cells (FIG. 5). All together these data indicate an advanced NK cell differentiation inducible by IL-12.

Ex Vivo with IL-12 Generated NK Cells Show Improved Adhesive Function on Lymphatic Endothelial Cells

[0073] The molecules involved in adhesion to lymphatic tissues, namely CD62L, and the chemokine receptors CCR1 and CCR6-8 allowing migration towards chemokine gradients thereby guiding migration into tissues, showed elevated expression on IL-12 modulated NK cells. Therefore, we performed in vitro assays to examine whether the IL-12 induced NK cell phenotype correlates with better adhesion in response to lymphoid tissues (FIG. 6). Assays comparing the adhesion to human umbilical vein endothelial cells (HUVEC) or lymphatic endothelial cells (LEC) showed that IL-12 induced ex vivo differentiated NK cells significantly better adhered to LEC than HUVEC cells, whereas not IL-12 modulated NK cells exhibited no enhanced adhesion to LEC compared to HUVEC cells (FIG. 6).

[0074] In summary these data reveal, that the modulation of NK cell differentiation by IL-12 leads to NK cells with improved adhesive that could exert certain migratory abilities potentially allowing increased homing to various tissues.

IL-12 Modified Ex Vivo Generated NK Cells Exert a Stronger Killing Capacity Towards AML Targets and Revealed Enhanced Antibody-Dependent-Cytotoxicity Reactions

[0075] Owing to the enhanced KIR expression and advanced differentiation stage of the IL-12 induced ex vivo generated NK cells we aimed to analyze if this might correlate with enhanced cytotoxicity in in vitro killing assays (FIG. 7). Assays combining an analysis of killing efficiency and CD107a activity against the MHC class I-negative, classical target cell line K562 and the MHC class I-positive cell line KG1a revealed better recognition and activity of the IL-12 modulated compared to not IL-12 induced ex vivo generated NK cells (FIGS. 7 A and B).

[0076] The enhanced CD16 expression of the NK cells differentiated under IL-12 modulation supposes an influence on their antibody-dependent-cytotoxicity (ADCC). The availability of therapeutical antibodies against many different human malignancies raised the question if the effect of these antibodies can be combined and enhanced with the cytotoxicity of the IL-12 modulated ex vivo generated NK cells. Hence, we compared the killing efficiency of ex vivo with or without IL-12 induction generated NK cells for their killing efficiency against several B-cell-lines pre-treated with the therapeutic B-cell-specific antibody Rituximab (FIG. 8). All B-cell lines tested, namely 721.221, REH, SEM, Nalm-6 and UoC-B6, were significantly better lysed by NK cells when they were pre-treated with Rituximab. Moreover, IL-12 modulated NK cells exhibited better killing capacities against all Rituximab coated B-cell lines than ex vivo generated NK cells not induced with IL-12. These data on the enhanced cytolysis of malignant target cells treated with therapeutic antibodies and ex vivo with IL-12 differentiated NK cells reveal a new aspect and functionality of this combination of therapeutic agents. Therefore, ex vivo differentiated IL-12 induced NK cells reveal a presumable therapeutic impact of these cells in combination with the various available therapeutical antibodies.

Discussion

[0077] The recently established ex vivo differentiation system for large scale generation of human NK cells holds great potential for adoptive immunotherapies of cancer.sup.14,15. Nevertheless, a tailored and modulated NK cell generation towards specified phenotypes and functions would facilitate the therapeutical use of these cells in an even broader range of malignancies. We have therefore analyzed several cytokines for their impact on the ex vivo NK cell differentiation and found IL-12 to be an especially strong modulator within this process. Under the influence of IL-12 during ex vivo NK cell differentiation the generated NK cells acquired higher expression of the cytotoxicity related KIR and CD16 receptors as well as CD62L and a specific chemokine receptor repertoire of CCR6, 7 and CXCR3-5 receptors related to homing and migration capacities of NK cells. Importantly, the optimized IL-12 concentration ensured the purity of the NK cell product whilst allowing an enhanced NK cell phenotype correlated with improved corresponding functions. Previous studies revealed the picture that IL-12 induced peripheral blood NK cells (PBNK) acquire CD56.sup.bright expression and exhibit mature and terminally differentiated NK cells, although discordant experimental findings led to this idea. On the one hand, IL-12 induced a CD56.sup.bright NK cell phenotype by up regulation of CD94 and CD62L and a down modulation of CD 16.sup.10. On the other hand, it was shown that CD 16.sup.CD56.sup.+ PBNK cells treated with IL-12 in combination with IL-2 and IL-15 developed CD 16 expression alongside with a CD56.sup.bright expression.sup.32. Nevertheless, these studies highlighted the impact of IL-12 on NK cell receptor expression and function. In contrast, others have dissected human NK cell subsets on the basis of CD56 and CD16 expression and suggested that CD56.sup.bright CD16.sup.+ NK cells represent an intermediate stage of NK cell maturation between CD56.sup.bright CD16.sup. and CD56.sup.dim CD 16.sup.+ NK cells already exhibiting full functional capacity.sup.33. Recently, we identified distinct stages of human NK cell development on the basis of CD33 and NKG2A expression.sup.31. Therefore, we can further strengthen the idea that IL-12 modulated ex vivo generated NK cells exhibit a more mature NK cell phenotype because of the increased proportion of CD33.sup.+NKG2A.sup.+ NK cells and, at least in cultures with especially high receptor induction, arising CD56.sup.dim NK cell subpopulation. Furthermore, whereas CD16 and KIR expression and the increased proportion of CD33.sup.+ NKG2A.sup.+ and CD56.sup.dim NK cells favour the idea of more mature NK cells, the induction of CD62L by IL12 during ex vivo NK cell differentiation is not contradictory. A recent study revealed that CD62L.sup.+CD56.sup.dim PBNK cells exhibit the full functional repertoire of NK cell cytokine production and cytotoxicity and are likely also representing an intermediate stage of NK cell differentiation towards cytotoxic CD56.sup.dim (CD 16.sup.+ KIR.sup.+) CD62L-NK cells.sup.34.

[0078] CD62L is an important receptor guiding NK cells into and out of lymph nodes through interactions with ligands on high endothelial venules and e.g. the ligand Mannose Receptor (MMR) along afferent and efferent lymphatic endothelium.sup.35,36. Moreover to adhesion molecules such as CD62L, specific chemokine receptors guide NK cells into lymphoid tissues and sites of tissue inflammation along chemotactic gradients. Therefore, also the induction of the CCR1, 6-7 and CXCR3-5 chemokine receptor repertoire on IL-12 induced ex vivo generated NK cells renders these cells with a potential of improved migratory functions, as we could already evidence in in vitro assays for the adhesion to lymphatic endothelial cells, which could be likely exploitable for therapies of lymphoid leukemia, lymphomas or solid tumors.

[0079] Early studies already indicated the potency of IL-12 to modulate the differentiation towards a cytotoxic and IFN- producing NK cell.sup.16. In recent years, studies evidenced these findings in patients with dysfunctions in IL-12-signaling pathways revealing the necessity of NK cell priming through IL-12 for the acquisition of functional activityl.sup.7. The acquisition of cytotoxic and IFN- producing NK cell functions by IL-12 was already correlated with induced expression of the IFN regulating factor-1 (IRF-1) and perforin genes.sup.18,19. In line with this, our in vitro killing assays against the MHC class I-positive KG1a and the MHC class I-negative K562 cell line confirmed an enhanced cytotoxic activity of the IL-12 induced ex vivo differentiated NK cells and support their possibly enhanced impact in antitumor therapies.

[0080] A characteristic of CD56.sup.dim NK cells is the ability to lyse antibody-coated target cells, a phenomenon named antibody-dependent-cytotoxicity (ADCC) which is mediated through the receptor CD16/FcRyIII. The enhanced expression of CD16 of ex vivo, under the influence of IL-12, differentiated NK cells might be utilized in therapeutic settings combining the cytotoxic activity of NK cells with therapeutic antibodies against malignant cells. Studies already revealed the potential and importance of e.g. the therapeutical antibody Rituximab recognizing CD20 on B-cell leukemias in combination with human PBNK cells.sup.37,38. This substantiates the improved functional capacity and potential therapeutic utilization of the IL-12 modulated ex vivo differentiated NK cells in combination with therapeutic antibodies, which we could already confirm in in vitro ADCC-assays against several B-cell lines coated with Rituximab antibodies.

[0081] Altogether, our findings indicate that IL-12 is an auspicious modulator of NK cell differentiation that can be exploited to generate NK cells with specified phenotypes and functions. This furthermore holds great potential and promise for the additional use of these cells in therapies of solid, especially lymphoid tumors and in combinational clinical settings accompanying therapeutic antibodies.

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