METHOD FOR OBTAINING CAR-NK CELLS

Abstract

The present invention relates to the field of manufacturing of Natural Killer (NK) Cells genetically modified with viral vectors carrying a polynucleotide coding for a Chimeric Antigen Receptors (CARs). The present invention further relates to CAR-NK cells obtained with the method and use of the CAR-NK cells in medicine, in particular for use in a method of treating cancer.

Claims

1-16. (canceled)

17. A method for the manufacturing of a population of cells, genetically modified with a Chimeric Antigen Receptor (CAR) comprising: a first step comprising: providing a sample comprising CD34+ hematopoietic stem cells purifying the CD34+ hematopoietic stem cells in said sample, culturing the purified CD34+ hematopoietic stem cells in the presence of culture medium I, transducing the purified CD34+ hematopoietic stem cells with a polynucleotide coding for a CAR by culturing the cellular populations in culture medium I in the presence of a vector comprising said polynucleotide for at least 10 hours, thereby obtaining a cellular population comprising CD34+ stem cells expressing said CAR, and culturing the cellular populations in culture medium I for at least 10 hours, wherein culture medium I is a basic culture medium, comprising a collection of cytokines, wherein said collection of cytokines comprises Interleukin-7 and one or more of stem cell factor (SCF), flt-3Ligand (FLT-3L), thrombopoietin (TPO), and two or more of granulocyte-macrophage-colony-stimulating factor (GM-CSF), granulocyte-colony-stimulating factor (G-CSF), and interleukin-6 (IL-6).

18. The method according to claim 17, wherein in step d) the cellular populations in culture medium I are cultured in the presence of a vector comprising said polynucleotide for at least 16 hours.

19. The method according to claim 17, wherein in step e) the cellular populations in culture medium I are cultured for at least 48 hours.

20. The method according to claim 17, wherein the cell culture of step c) is initiated at a cell density of between 500 and 10,000 CD34.sup.+ cells/ml.

21. The method according to claim 20, wherein the cell culture of step c) is initiated at a cell density of between 1,000 and 8,000 CD34+ cells/ml.

22. The method according to claim 17, wherein the polynucleotide does not encode for a CAR specific for an antigen expressed on hematopoietic stem cells, natural killer (NK) progenitor cells or NK cells.

23. The method according to claim 17, further comprising: a second step in which the cellular population from step (i) is expanded and differentiated into a cellular population containing CAR-NK progenitor cells or CAR-NK cells or both, the step comprising culturing the cellular population from step (i) containing CAR-CD34+ stem cells in culture medium III, thereby obtaining a cellular population containing CAR-NK progenitor cells or CAR-NK cells or both, wherein the culture medium III is a basic culture medium comprising a collection of cytokines, wherein said collection of cytokines comprises two or more of SCF, IL-7, IL-15 and interleukin-2 (IL-2) and two or more of GM-CSF, G-CSF, and IL-6.

24. The method according to claim 17, further comprising: a second step in which the cellular population from step (i) is expanded and differentiated into a cellular population containing CAR stem cells or CAR progenitor cells or both, the step comprising culturing the CAR-CD34+ stem cells from step (i) in culture medium II, thereby obtaining a cellular population containing CAR stem cells or CAR progenitor cells or both, and a third step in which the cellular population from step (ii) is further expanded and differentiated into a cellular population containing CAR-NK progenitor cells or CAR-NK cells or both, wherein culture medium II is a basic culture medium comprising a collection of cytokines, wherein said collection of cytokines comprises two or more of SCF, FLT-3L interleukin-15 (IL-15) and IL-7 and two or more of GM-CSF, G-CSF, and IL6, and wherein culture medium III is a basic culture medium comprising a collection of cytokines, wherein said collection of cytokines comprises two or more of SCF, IL-7, IL-15 and interleukin-2 (IL-2) and two or more of GM-CSF, G-CSF, and IL-6.

25. The method according to claim 17, wherein the sample has been obtained from umbilical cord blood.

26. The method according to claim 17, wherein stem cells are purified using CD34+ immunomagnetic selection methods.

27. The method according to claim 17, wherein culture medium I comprises SCF at concentration between 4 ng/ml and 300 ng/ml, or Flt3-L at concentration between 4 ng/ml and 300 ng/ml, or TPO at concentration between 4 ng/ml and 100 ng/ml, or IL7 at concentration between 4 ng/ml and 50 ng/ml, or any combination of these cytokines in the specified concentrations.

28. The method according to claim 17, wherein CD34+ stem cells are incubated with a viral vector containing said polynucleotide coding for said CAR at multiplicity of infection (MOI) between 0.01 and 100

29. The method according to claim 28, wherein CD34+ stem cells are incubated with a viral vector containing said polynucleotide coding for said CAR at multiplicity of infection (MOI) between 1 and 10.

30. The method according to claim 17, wherein the viral vector is a retroviral vector.

31. The method according to claim 17, wherein the CAR is directed against a tumour antigen.

32. A composition comprising an NK-CAR cell obtainable by a method according to claim 17.

33. The composition according to claim 32, wherein the NK-CAR cell is positive for Neural Cell Adhesion Molecule (NCAM) and a CAR.

34. A method for treating a subject having a tumour or a haematological malignancy, comprising administering to the subject a composition according to claim 32.

Description

DESCRIPTION OF THE DRAWINGS

[0124] FIG. 1: We designed a modular CAR molecule that allows easy incorporation of novel scFv proteins via a single cloning procedure. The FseI-SbfI-flanked stuffer fragment allows easy seamless cloning of targeting molecules into the empty CAR backbone. As shown in FIGS. 1b and c, we inserted scFv proteins into this space which target the transduced NK cell to respectively BCMA+ or EGFR+ tumor cells. FIG. 1d illustrates a control vector that contains an irrelevant scFv that has no binding affinity to any human protein. The IgD hinge region confers the flexibility to the CAR, while the optimized IgG1 heavy chain sequence functions as spacer region. The CAR is anchored into the membrane via de CD28 transmembrane sequence. This 3.sup.rd generation CAR contains the co-stimulatory domains of CD28 and OX-40 and the activation domain of CD3zeta. FIG. 1e shows the location of each domain with respect to the cell membrane. Arrows indicate unique restriction sites that can be used to incorporate scFvs (via FseI and SbfI), transfer the CAR into a lentiviral transfer plasmid (arrows on either side of the CAR) or to change co-stimulatory or activation domains (arrows flanking each domain)

[0125] FIG. 2: Representative example of flow cytometry data from two different culture conditions using two donors. Viable cells are gated on CD45+ lymphocytes. The figure illustrates the expression of CD44v6 on NK cells (A) and progenitor cells (B) if hematopoietic stem and progenitor cells are expanded and differentiated according to technology described in this invention. A. CD44v6 expression of day 29 oNKord cells (% CD56: 92%), B. CD44v6 expression on day 14 oNKord progenitor cells (% CD56: 1.2%)

[0126] FIG. 3: Transduction efficiency in CD34+ cells. CD34+ HSC cells were transduced with VSV-G pseudotyped lentiviral particles containing EGFP at MOI 20. Cells were expanded in expansion/differentiation medium for subsequent 29 days and the percentage of EGFP positive cells was determined by flow cytometry. Three different donors were used for the transduction, measurements were performed in triplicates, mean±SD is shown.

[0127] FIG. 4: Transduction of precursors derived from CD34+ HSC. Precursor cells derived from CD34+ HSC (NK) were transduced with VSV-G pseudotyped lentiviral particles containing EGFP at MOI 20 after 21, 28 and 34 days in culture in expansion/differentiation medium. Cells were maintained in differentiation medium for subsequent 6 days after transduction and the percentage of EGFP positive cells was determined by flow cytometry. Three different donors were used for the transduction, measurements were performed in duplicates, mean of three different donors±SD is shown.

EXAMPLES

Example 1: Generation of a 3.SUP.rd .Generation CAR Construct

[0128] The CAR construct used in these examples is synthetically generated by ID&T DNA technologies as a single polynucleotide with flanking restriction sites that allow easy transfer into a suitable expression vector. The CAR expression cassette is fully human codon optimized for efficient expression in human cells. The vector consists of a CD8a signal peptide, an FseI-SbfI flanked stuffer fragment, the IgD hinge region, the IgG1 heavy constant fragment optimized to avoid binding to IgG Fc gamma receptors and thus to inhibit unintentional activation of innate immune cells (Hombach et al., Gene Therapy, 2010), the CD28 transmembrane domain, the CD28 co-activation domain, the OX40 co-activation domain and the CD3zeta activation domain. Each (co-) activation domain is flanked by unique restriction sites that allows testing of each individual domain.
A schematic version of this CAR cassette is shown in FIG. 1.
The protein sequence of this CAR cassette is shown below:

TABLE-US-00002 CD8α leader sequence: MDALPVTALLLPLALLLHAEVQLQQS FseI-SbfI flanked staffer fragment: GRPSLSESCRA IgD hinge region: AFRWPESPKAQASSVPTAQPQAEGSL Optimized IgG1 heavy chain Fc part, optimized: AKATTAPATTRNTGGRGGEEKKKEKEKEEQEERETKTPEAAAVEPKSCD KTHTCPPCAAPPVAGPSVFLFPPKPKDTLMIARTPEVTCVVVDVSHEDP EVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYK CKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLV KGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQ QGNVFSCSVMHEALHNHYTQKSLSLSPGK CD28 transmembrane domain: FWVLVVVGGVLACYSLLVTVAFIIFWV CD28 co-activation domain: RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS OX40 co-activation domain: RRDQRLPPDAHKPPGGGSFRTPIQEEQADAHSTLAKI CD8zeta activation domain: RVKFSRSAEPPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKP RRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATK DTYDALHMQALPPR
To generate specific CARs against BCMA (for the treatment of multiple myeloma) and EGFR (for the treatment of metastatic colorectal cancer, metastatic non-small cell lung cancer and head and neck cancer), we substitute the stuffer fragment for single-chain variable fragment protein sequences against BCMA and EGFR by seamless cloning.

TABLE-US-00003 BCMA (source: patent US 2016/0046724 A1): EVQLVESGGGLVQPGGSLRLSCAVSGFALSNHGMSWVRRAPGKGLEWVS GIVYSGSTYYAASVKGRFTISRDNSRNTLYLQMNSLRPEDTAIYYCSAH GGESDVWGQGTTVTVSSASGGGGSGGRASGGGGSDIQLTQSPSSLSASV GDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSG SGSGTDFTLTISSLQPEDFATYYCQQSYSTPYTFGQGTKVEIK EGFR (source: cetuximab antibody sequence): QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLG VIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARA LTYYDYEFAYWGQGTLVTVSAGGGGSGGGGSGGGGSDILLTQSPVILSV SPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRF SGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLELKR
The entire CAR cassette (including relevant ScFv sequence) is inserted into a 3.sup.rd generation lentiviral backbone plasmid via unique restriction sites flanking the CAR cassette. In this vector, the EF1alpha promoter drives the constitutive expression of the CAR polynucleotide.
Viral vectors are produced in HEK293T cells by transfection of 3.sup.rd generation helper plasmids in combination with the selected lentiviral transfer plasmid containing a polynucleotide encoding the CAR. Twenty-four hours post-transfection, cells are replenished with a basal culture medium supplemented with 10% FBS and incubated at 5% CO.sub.2 and 37 degrees Celsius. Forty-eight hours later, the first harvest of viral vectors is performed and the producer cells are replenished with the medium mentioned above. Another 24 hours later, the second, and final, harvest is performed.
Next, both harvests are combined, filtered through a 0.22 um or 0.45 um PES filter and treated with benzonase (50 U/ml) to remove any of the remaining plasmid DNA. Next, viral supernatant is concentrated by PEG6000 precipitation. Briefly, viral supernatant is mixed with 4× Precipitation Buffer (consisting of 0.5M NaCl and 7.5% PEG6000) and centrifuged for 20 minutes at 7000×g at 4 degrees Celsius. Next, the supernatant is removed and resuspended in the appropriate culture medium and used immediately for downstream transduction or stored at −80 degrees Celsius for later use.
Here, we distinguish 4 different growth media:

TABLE-US-00004 Basal Medium Additional cytokines MEDIUM A CellGenix ® GMP 100 ng/ml Flt3-L, 100 ng/ml Stem cell Growth SCF, 50 ng/ml TPO Medium and 50 ng/ml IL-3 MEDIUM B CellGenix ® GMP 100 ng/ml Flt3-L, 100 ng/ml Stem cell Growth SCF, 50 ng/ml TPO Medium and 50 ng/ml IL-6 MEDIUM C Glycostem Basal 100 ng/ml Flt3-L, 100 ng/ml Growth Medium SCF, 50 ng/ml TPO and 50 ng/ml IL-7 MEDIUM D Glycostem Basal 25 ng/ml Flt3-L, 25 ng/ml Growth Medium SCF, 25 ng/ml TPO and 25 ng/ml IL-7, 10% human serum, 10 pg/ml GM-CSF, 250 pg/ml G-CSF and 50 pg/ml IL6

Example 2: Transduction of CD34.SUP.+ Hematopoietic Stem Cells by Lentiviral Vector

[0129] During the first phase of the method of the invention, CD34+ stem cells are isolated from starting material by immunomagnetic separation technique and subsequently transduced by lentiviral vector containing one or more polynucleotides encoding a CAR, yielding CAR-NK cells. The goal is to obtain a stable population of CAR-NK cells that are expanded in the second phase of the method of the invention.

Starting Material

[0130] The starting material to be used in the method of the present invention is a biological sample containing adult (postembryonic) stem cells, also called somatic stem cells. As used herein, the term biological sample means a sample derived from a human being. In a preferred embodiment, the starting material to be used is umbilical cord blood.

Isolation of CD34+ Stem Cell Population

[0131] CD34+ stem cells are isolated from umbilical cord blood via the Miltenyi Prodigy® closed-system cell manufacturing platform. Briefly, cells are purified by magnetic separation via CD34-conjugated magnetic beads. The end product is a pure CD34+ population that is collected in culture medium I and is either used for expansion and/or differentiation, transduction or frozen down in a freezer via a controlled temperature profile.

Transduction of CD34+ Cells to Obtain a CAR-NK Population

[0132] Immediately following isolation or thawing, CD34+ stem cells are counted and immediately resuspended in the viral stock prepared in example 1 at an MOI of 1-50 and plated in Retronectin-coated plates. Optionally, transduction efficiency may be enhanced by the addition of 10 ug/ml polybrene or 10 ug/ml protamine sulfate.
Including control cells, we distinguish:

TABLE-US-00005 Cells Medium MOI Condition CD34+ MEDIUM A 1 CAR-T vector containing scFV against BCMA CD34+ MEDIUM A 10 CAR-T vector containing scFV against BCMA CD34+ MEDIUM A 50 CAR-T vector containing scFV against BCMA CD34+ MEDIUM A 1 CAR-T vector containing scFv against EGFR CD34+ MEDIUM A 10 CAR-T vector containing scFv against EGFR CD34+ MEDIUM A 50 CAR-T vector containing scFv against EGFR CD34+ MEDIUM A 1 CAR-T vector containing irrelevant scFV (CAR-NK-IRR) CD34+ MEDIUM A 10 CAR-T vector containing irrelevant scFV (CAR-NK-IRR) CD34+ MEDIUM A 50 CAR-T vector containing irrelevant scFV (CAR-NK-IRR) CD34+ MEDIUM A N/A MOCK CD34+ MEDIUM B 1 CAR-T vector containing scFV against BCMA CD34+ MEDIUM B 10 CAR-T vector containing scFV against BCMA CD34+ MEDIUM B 50 CAR-T vector containing scFV against BCMA CD34+ MEDIUM B 1 CAR-T vector containing scFv against EGFR CD34+ MEDIUM B 10 CAR-T vector containing scFv against EGFR CD34+ MEDIUM B 50 CAR-T vector containing scFv against EGFR CD34+ MEDIUM B 1 CAR-T vector containing irrelevant scFV (CAR-NK-IRR) CD34+ MEDIUM B 10 CAR-T vector containing irrelevant scFV (CAR-NK-IRR) CD34+ MEDIUM B 50 CAR-T vector containing irrelevant scFV (CAR-NK-IRR) CD34+ MEDIUM B N/A MOCK CD34+ MEDIUM C 1 CAR-T vector containing scFV against BCMA CD34+ MEDIUM C 10 CAR-T vector containing scFV against BCMA CD34+ MEDIUM C 50 CAR-T vector containing scFV against BCMA CD34+ MEDIUM C 1 CAR-T vector containing scFv against EGFR CD34+ MEDIUM C 10 CAR-T vector containing scFv against EGFR CD34+ MEDIUM C 50 CAR-T vector containing scFv against EGFR CD34+ MEDIUM C 1 CAR-T vector containing irrelevant scFV (CAR-NK-IRR) CD34+ MEDIUM C 10 CAR-T vector containing irrelevant scFV (CAR-NK-IRR) CD34+ MEDIUM C 50 CAR-T vector containing irrelevant scFV (CAR-NK-IRR) CD34+ MEDIUM C N/A MOCK CD34+ MEDIUM D 1 CAR-T vector containing scFV against BCMA CD34+ MEDIUM D 10 CAR-T vector containing scFV against BCMA CD34+ MEDIUM D 50 CAR-T vector containing scFV against BCMA CD34+ MEDIUM D 1 CAR-T vector containing scFv against EGFR CD34+ MEDIUM D 10 CAR-T vector containing scFv against EGFR CD34+ MEDIUM D 50 CAR-T vector containing scFv against EGFR CD34+ MEDIUM D 1 CAR-T vector containing irrelevant scFV (CAR-NK-IRR) CD34+ MEDIUM D 10 CAR-T vector containing irrelevant scFV (CAR-NK-IRR) CD34+ MEDIUM D 50 CAR-T vector containing irrelevant scFV (CAR-NK-IRR) CD34+ MEDIUM D N/A MOCK
Optionally, 24 hours post-transduction, cells are washed with the selected medium and are re-transduced at the same MOI. Following 24 hours of incubation, cells are replenished with the culture medium II.

Example 3: Expansion and Differentiation of CAR-CD34+ Cells

Cell Culture

[0133] The transduced cells of condition A-D are cultured directly, or if cryopreserved CAR transduced and MOCK progenitor cells from condition A-D are thawed in thawing buffer consisting of human serum albumin supplemented with 2.5 mM MgCl2 and 0.13 mg/ml DNAse. The CD34+ UCB cells are plated in tissue culture treated 6-wells plates in fresh culture medium I at cell concentrations of 0.02-2×10{circumflex over ( )}6 cells/ml for conditions A-D.

Expansion Phase

[0134] Flow cytometry data for cell viability, CD34 content and CAR expression are measured in the expansion phase to monitor and provide optimal cell culture conditions. CD34+ UCB cells are cultured in fresh culture medium I consist of GBGM supplemented with 10% human serum, a low dose cytokine cocktail containing 10 pg/ml GM-CSF, 250 pg/ml G-CSF and 50 pg/ml IL-6; 25 ng/ml SCF, Flt-3L, TPO, IL-7. Cells from condition A-D are cultured in culture medium I till day 9. Culture medium I is refreshed every 2-3 days a week. At day 10, the progenitor cells are cultured by adding culture medium II, hereby replacing 25 mg/ml TPO for 20 ng/ml IL-15.

Differentiation Phase

[0135] During differentiation, the CD56 content as a marker of NK-cells is measured instead of the CD34 content alongside cell viability and CAR expression. Differentiation medium (aka culture medium III) consists of GBGM supplemented with 2% human serum, a low dose cytokine cocktail containing 10 pg/ml GM-CSF, 250 pg/ml G-CSF and 50 pg/ml IL-6; 20 ng/ml SCF, IL-15, IL-7 and 1000 U/ml IL-2 (Proleukin) Differentiation medium is refreshed twice a week until end of culture.
The CAR expression in the BCMA and EGFR transduced UCB-NK cells reveals relative high CAR expression in condition A-C transduced NK-cells and mediate expression in condition D transduced NK-cells.

Example 4: Anti-Tumor Potential of CAR-NK-BCMA and CAR-NK-EGFR Cells Versus Control CAR-NK-IRR and MOCK Cells

[0136] An in vitro functionality assay was performed at the end of the culture with Mock and CAR transduced UCB-NK cells from condition A-D against a battery of multiple myeloma and colorectal cancer cell lines (see table below) showing sensitive-, intermediate- and resistance to NK-cell cytotoxicity.

TABLE-US-00006 Myeloma Colorectal Cancer Cell lines Cell lines RPMI-8226 HT-29 U-266 TC71 OPM-2 HCT-15 LP-1 SW480 SK-MM2 COLO320 NCI-H929 RKO
Target cells (see table above) were labeled with 5 μM pacific blue succinimidyl ester (PBSE) at a concentration of 1×10{circumflex over ( )}7 cells/ml for 10 minutes at 37° C. The target cells were washed in target culture medium and concentrated to 5×10{circumflex over ( )}5 cells/ml. The NK-cells were concentrated to 5×10{circumflex over ( )}5 cells/ml as well and co-cultured with target cells (100 μl effectors+100 μl targets) in an overnight assay. For degranulation measurements anti-CD107a was added at the start of the incubation and anti-CD56 for NK-cell discrimination at the end of the incubation. Cytotoxicity was calculated based on flow cytometry read out for the apoptotic 7AAD viability marker for Effector:Target (E:T) ratios of 1:1.
Lower cytotoxicity is observed for the BCMA and EGFR transduced UCB-NK cells from condition A-C against the sensitive tumour cell lines (see table above) compared to condition D.

Example 5: Expansion and Differentiation of CD34+ Cells

Cell Culture

[0137] CD34+ UCB cells are plated in tissue culture treated 6-wells plates in fresh culture medium I at cell concentrations of 0.02-2×10{circumflex over ( )}6 cells/ml for conditions A-D.

Expansion Phase

[0138] Flow cytometry data for CD45 and CD56 as well as CD44v6 are measured in the expansion phase to monitor and provide optimal cell culture conditions. CD34+ UCB cells are cultured in fresh culture medium I consist of GBGM supplemented with 10% human serum, a low dose cytokine cocktail containing 10 pg/ml GM-CSF, 250 pg/ml G-CSF and 50 pg/ml IL-6; 25 ng/ml SCF, Flt-3L, TPO, IL-7. Cells are cultured in culture medium I till day 9. Culture medium I is refreshed every 2-3 days a week. At day 10, the progenitor cells are cultured by adding culture medium II, hereby replacing 25 mg/ml TPO for 20 ng/ml IL-15. At day 14 cell culture is analysed for CD56 NK cell content and CD44v6 expression as shown in FIG. 2.

Differentiation Phase

[0139] During differentiation, the CD56 content as a marker of NK-cells is measured together with CD45 and CD44v6 expression. Differentiation medium (aka culture medium III) consists of GBGM supplemented with 2% human serum, a low dose cytokine cocktail containing 10 pg/ml GM-CSF, 250 pg/ml G-CSF and 50 pg/ml IL-6; 20 ng/ml SCF, IL-15, IL-7 and 1000 U/ml IL-2 (Proleukin). Differentiation medium is refreshed twice a week until end of culture. At day 29 cell culture is analysed for CD56 NK cell content and CD44v6 expression as shown in FIG. 2.

Example 6: Transduction of CD34.SUP.+ Hematopoietic Stem Cells and Precursor Cells Derived from CD34+ Hematopoietic Stem Cells by Lentiviral Vector

[0140] CD34+ stem cells are isolated from starting material by immunomagnetic separation technique and subsequently transduced with a lentiviral vector, pseudotyped by a VSVG envelop, containing one or more polynucleotides encoding an EGFP, yielding EGFP-NK cells (pLenti CMV GFP Puro (658-5); Addgene plasmid #17448; n2t.net/addgene:17448; RRID:Addgene_17448; Campeau E, Ruhl V E, Rodier F, Smith C L, Rahmberg B L, Fuss J O, Campisi J, Yaswen P, Cooper P K, Kaufman P D. PLoS One. 2009 Aug. 6; 4(8):e6529). Alternatively, CD34+ stem cells are cultured as described previously (Method III in Spanholtz J, Tordoir M, Eissens D, Preijers F, van der Meer A, Joosten I, et al. PLoS ONE 2010 5(2): e9221) to achieve precursor cells; and these precursor cells are transduced with the above lentiviral vector at day 21, 28, or 34, before further expansion and differentiation.

Starting Material

[0141] Here, we distinguish 2 different growth media:

TABLE-US-00007 Basal Medium Additional cytokines MEDIUM A Glycostem Basal 50 ng/ml Flt3-L, 50 ng/ml Growth Medium SCF, 50 ng/ml TPO 50 ng/ml IL-7, 20 pg/ml GM-CSF, 500 pg/ml G-CSF and 100 pg/ml IL6 MEDIUM B Glycostem Basal 20 ng/ml SCF, 20 ng/ml Growth Medium IL-7, 2% human serum, 10 pg/ml GM-CSF, 250 pg/ml G-CSF, 50 pg/ml IL6, 25 ng/mL IL-7, 20 ng/mL IL-15, 20 ng/mL SCF, 1000 U/mL IL-2

Isolation of CD34+ Stem Cell Population

[0142] CD34+ stem cells are isolated from umbilical cord blood via the Miltenyi Prodigy® closed-system cell manufacturing platform (Miltenyi Biotec B.V.&Co KG, Bergisch Gladbach, Germany). Briefly, cells are purified by magnetic separation via CD34-conjugated magnetic beads. The end product is a pure CD34+ population that is collected in culture medium I and is either used for expansion and/or differentiation, or frozen down in a freezer via a controlled temperature profile.

Transduction of CD34+ Cells or Precursors Derived Thereof to Obtain an EGFP-NK Population

[0143] Immediately following thawing, CD34+ stem cells resuspended in Medium A. Twenty-four hours later cells are counted, resuspended at a concentration of 5,000 cells per 100 μl of fresh Medium A per well in 96-well plates. Viral stock prepared in example 1 was diluted in Medium A at an MOI of 20 per 100 μl in Retronectin-coated plates (Takara Bio Europe SAS, St Germain en Laye, France). Cell were added to the plates subsequently. Optionally, transduction efficiency may be enhanced by the addition of following transduction enhancers to the viral stock to reach these final concentrations in Medium A: 8 μg/ml polybrene (Sigma-Aldrich Chemie N.V., Zwijndrecht, The Netherlands), 4 μ/ml protamine sulfate (Sigma-Aldrich Chemie N.V.), 1 μg/ml Vectofusin®-1 (Miltenyi Biotec B.V.&Co KG), 4 μ/ml protamine sulfate plus 1 μg/ml Vectofusin®-1, 1 mg/ml LentiBOOST™ (Sirion Biotech GmbH, Martinsried, Germany), or 1 μl/100 μl media of LentiBlast Premium (OZBiosciences SAS, Marseille, France).
Alternatively, CD34+ cells are cultured in expansion and differentiation medium and resulting precursor cells are transduced as described above. Transduction was performed with cells resuspended at a concentration of 200,000 cells per 250 μl of fresh Medium B per well in 24-well plates. Viral stock prepared in example 1 was diluted in Medium B at an MOI of 20 per 250 μl. Transduction enhancers were used at the same final concentrations as described above.
Including control cells, we distinguish:

TABLE-US-00008 Cells Medium MOI Condition CD34+ MEDIUM A 20 EGFP vector CD34+ MEDIUM A N/A MOCK Precursors derived from CD34+ cells at day 21 MEDIUM B 20 EGFP vector Precursors derived from CD34+ cells at day 21 MEDIUM B N/A MOCK Precursors derived from CD34+ cells at day 28 MEDIUM B 20 EGFP vector Precursors derived from CD34+ cells at day 28 MEDIUM B N/A MOCK Precursors derived from CD34+ cells at day 35 MEDIUM B 20 EGFP vector Precursors derived from CD34+ cells at day 35 MEDIUM B N/A MOCK
Following 24 hours of incubation, CD34+ cells are replenished with the culture medium II. Following 24 hours of incubation, precursors derived from CD34+ cells are replenished with the culture medium III.