1. Clinical Scale Generation of Functional Human Natural Killer Cells From Umbilical
Cord Blood CD34-Positive Cells for Immunotherapy
Jan Spanholtz, Marleen Tordoir, Carel Trilsbeek, Jos Paardekooper, Theo de Witte, Nicolaas Schaap, Frank Preijers and Harry Dolstra
Department of Laboratory Medicine - Laboratory of Hematology, Radboud University Nijmegen Medical Centre, The Netherlands
mailto: j.spanholtz@labgk.umcn.nl
Cell processing using a closed system allows the GMP Efficient NK cell production from cryopreserved UCB units Ex-vivo generated NK cells could be efficiently processed and released as
generation of NK cells from cryopreserved umbilical cord blood using various bioreactors (step 3+4) “UCB-NK cell therapy product” (step 5+6)
A
a Bb Donor 7 Donor 8 Donor 9 Donor 7 Donor 8 Donor 9
A B C
Fold expansion total cells
Step 1. Thawing & washing UCB Step 2. CD34 CliniMACS enrichment 10.000 100
CD 56+ cells (%)
80
120 WBC (n=3) NK cells (n=3)
 thawing at 37oC  adding CD34 reagent and Nanogam 1.000 bioreactor cultures (n=3)
bioreactor cultures (n=3)
dilution in buffer with DNAse  incubation 30 min at RT 60 100 bioreactor cultures washed (n=3)
bioreactor cultures washed (n=3)
 incubation 30 min at RT  washing & centrifugation 100
recovery (%)
specific lysis (%)
40
Static bag cultures 80
 washing & centrifugation  docking cell bag to tubing set 80 30
CD107a+ cells (%)
 resuspension in CliniMACS buffer  performing automated program 10 20 60
 collecting CD34+ fraction 60
20
1 0 40
0 1 2 3 4 5 6 40
0 1 2 3 4 5 6
20 10
Culture duration (weeks) Culture duration (weeks) 20
Frozen UCB Thawed UCB
C
c
Donor 10 Donor 13 Donor 15 Donor 16
Dd Donor 10 Donor 13 Donor 15 Donor 16 0 0
0
Fold expansion total cells
10.000 100
Bag Tube E:T 1:1 E:T 10:1 E:T 1:1 E:T 10:1
CD 56+ cells (%)
80
1.000
Ex-vivo generated NK cells were washed in bags and NK cells were efficiently
60
Step 3. CD34+ cell expansion Step 4. NK cell differentiation 100 WAVE/Biostat cultures recovered (A) without the loss of function (B+C). NK cell products can be stored for
40
Day 0-14 Day 14-42 10
72hrs in infusion buffer (D). The products showed genetic stability (no changes in
20
CD34+ UCB cells NK progenitors Mature NK cells karyotype analyzes) and were negative for mycoplasm and sterility tests. The
1 0
In-process controls 0 1 2 3 4 5 6 0 1 2 3 4 5 6 detailed release criteria are listed in table E.
 sterility Culture duration (weeks) Culture duration (weeks)
 purity
 viability Ex-vivo generated NK cells are highly pure (≥ 90%) and contain D Table: Storage conditions and duration of UCB-NK-cell products.
up to 3.7x109 NK cells generated from 1.5x106 CD34+ cells UCB-NK-cell product Donor10 UCB-NK-cell product Donor16
GBGM + 10% HS GBGM + 10% HS
SCF, Flt3L, IL-7, TPO or IL-15 (high dose) SCF, Flt3L, IL-7, IL-15, IL-2 (high dose) Bioreactor Donor CD34+ cells (x106) fold expansion CD56+ cells (%) CD56+ cells (x109) Volume end-product 87 ml 97 ml
IL-6, G-CSF, GM-CSF (low dose) IL-6, G-CSF, GM-CSF (low dose)
clinical grade heparin Static bag 7 1.7 1770 63 1.9 Viability directly after washing 94% 88%
Static bag 8 1.4 759 80 0.9
Static bag 9 1.3 1291 70 1.2 Viability after 24 hr RT: 86% and 4oC: 85% RT: 84% and 4oC: 88%
WAVE/Biostat 10 0.9 2549 95 2.2
Viability after 48 hr RT: 81% and 4oC: 83% RT: 78% and 4oC: 81%
Step 5. Centrifugation & Washing Step 6. Quality control testing WAVE/Biostat 13 1.5 1764 90 2.4
WAVE/Biostat 15 1.5 2657 92 3.7 Viability after 72 hr RT: 84% and 4oC: 82% RT: 76% and 4oC: 72%
 centrifugation  sterility WAVE/Biostat 16 1.2 1435 92 1.6
Recovery directly after washing 83% 76%
 washing 2x with 500 ml buffer  mycoplasm
 resuspension in 150-300 ml infusion buffer  phenotype Ex-vivo generated NK cells are devoid of T- and B cells efficiently Recovery after 24 hr RT: 100% and 4oC: 100% RT: 100% and 4oC: 100%
 purity
 B and T cell content
lyse various primary AML tumor cells
Recovery after 48 hr RT: 80% and 4oC: 80% RT: 100% and 4oC: 100%
 Absence cytokines E Recovery after 72 hr RT: 80% and 4oC: 76% RT: 70% and 4oC: 88%
Washed NK cell product UCB-NK-cell product
95 0 91 0
5 0 9 0
Table: Specifications end-product controls NK cell product.
E
CD56
CD38
CD45
Test of Method Laboratory site Release criteria
Dept. Microbiology, Negative for bacterial and fungal
Sterility Culture
RUNMC contamination
GMP clinical scale generation of allogeneic NK cell products Luminescence Lab. of Hematology,
Mycoplasm Negative for mycoplasm contamination
assay RUNMC
Lab. of Hematology, Positivity for CD56, CD94, NKG2A, NCR and
Efficient CD34+ cell enrichment from cryopreserved UCB units F
SSC CD3 CD19 Phenotype Flow cytometry
RUNMC NKG2D
using the CliniMACS system (step 1+2) day1 E:T ratio 3:1
Purity Flow cytometry
Lab. of Hematology,
RUNMC
>70% CD56+CD3- NK cells
day2
specific lysis (%)
100 Lab. of Hematology,
day3 Viability Flow cytometry >70% 7-AAD negative
Calculation on nucleated cells (AcT10 counter) Calculation on CD45+7AAD- cells using beads RUNMC
80
CD34 Lab. of Hematology, < 1x104 CD3+ T cells/kg body weight of the
CD34+ positive fraction WBC CD34 recovery recovery spCD34 recovery recovery T cell content Flow cytometry
content 60 RUNMC patient
x106 x106 total % CD34 sel % x106 total % CD34 sel % CD34% Lab. of Hematology, < 1x104 CD19+ B cells/kg body weight of
40 B cell content Flow cytometry
RUNMC the patient
Donor 1 1.80 0.93 22 29 1.47 38 50 52
20 Absence of Lab. of Hematology,
Donor 2 3.00 2.32 37 54 1.99 34 53 77 ELISA < 25 pg/ml IL-2, IL-7, IL-15 and SCF
cytokines RUNMC
Donor 3 3.00 2.11 43 69 2.36 48 73 70 0
AML1 AML2 AML3 AML4 AML5 K562 KG1a
Donor 4 7.30 6.73 61 79 6.34 73 78 92
Donor 5 2.40 1.30 35 62 1.74 47 76 54
Donor 6 2.50 1.63 45 65 1.70 54 79 65 Adoptive transfer of CD34+ derived NK cells in poor-prognosis AML patients
Donor 7 2.30 1.47 65 74 1.70 82 82 64 Clinical protocol (phase I/II study)
Donor 8 1.70 1.24 43 63 1.42 53 69 73 UCB banks HLA – haploidendical donor
sibling or child >18 years
AML patient >65 years
treated with remission-induction chemotherapy
 Glycostem® clinical grade NK cell generation system provides sufficient numbers of
Donor 9 1.50 1.32 35 51 1.32 33 72 88
Donor 10 1.30 0.89 53 70 n.d. n.d. n.d. 69 Complete remission
functional NK cells from UCB CD34+ cells
(<5% blasts in bone marrow)
Donor 11
Donor 12
2.90
4.20
1.89
2.48
43
31
80
42
2.29
2.79
81
40
91
70
65
59
HLA and KIR typing  Process is transferred into a GCP standard operating procedure.
Donor 13 1.96 1.64 48 68 1.47 48 55 84 KIR-ligand incompatibility Consolidation
chemotherapy  Clinical protocol (phase I/II) has been designed for testing safety and toxicity
Donor 14 1.50 1.01 30 69 1.09 62 76 67
 NK cells as Donor Lymphocyte Infusion (DLI) for AML patients in non transplantation
CD34 selection(BM or UCB)
Donor 15 2.40 1.05 35 52 1.52 63 71 44
Two-step protocol
Donor 16 2.10 1.10 39 61 1.19 43 65 52
mean 2.62 1.82 41 62 2.03 53 71 67
ex vivo NK cell expansion
(3x108 -1x1010 NK cells)
Non-myeloablative immunosuppression
Flu 30 mg/m2 and Cy 1200 mg/m2 at day -6, -5, -4, -3 setting
st.dev 1.45 1.40 11 14 1.28 16 11 14 NK cell infusions(escalating doses)
I. 3x106 NK cells/kg (3 patients)  Escalating doses will be applied in KIR-ligand mismatched setting
median 2.35 1.40 41 64 1.70 48 72 66 II.10x106 NK cells/kg (3 patients)
III. 3x107 NK cells/kg (3 patients)
min 1.30 0.89 22 29 1.09 33 50 44 IV.10x107 NK cells/kg (3 patients)
max 7.30 6.73 65 80 6.34 82 91 92
Evaluation for
Toxicity, NK cell survival and expansion, disease status, GVL effect
 GBGM® can be purchased via www.glycostem.nl