Rapid generation of clinical-grade antiviral T cells: selection of suitable T-cell donors and GMP-compliant manufacturing of antiviral T cells

Tischer, Sabine; Priesner, Christoph; Heuft, Hans-Gert; Goudeva, Lilia; Mende, Wolfgang; Barthold, Marc; Kloeß, Stephan; Arseniev, Lubomir; Aleksandrova, Krasimira; Maecker-Kolhoff, Britta; Blasczyk, Rainer; Koehl, Ulrike; Eiz-Vesper, Britta

doi:10.1186/s12967-014-0336-5

Table 2 Outcome of CMVpp65-specific T-cell separation by CliniMACS CCS

From: Rapid generation of clinical-grade antiviral T cells: selection of suitable T-cell donors and GMP-compliant manufacturing of antiviral T cells

A	1. Validation run
A	Leukapheresis	OF	TCF	WF	NF
volume [ml]	133	100	40	335	276
viability [%]	99.80	99.06	51.13	92.34	99.38
WBCs_(CD45+) [x10⁶/ml]	6.77	1.42	0.14	0.02	0.49
WBCs_abs [x10⁶]	900.41	142.00	5.51	8.15	136.07
T cells_(CD3+CD56-) [% of WBCs]	41.93	47.80	31.68	41.58	44.55
T cells [/μl]	2834.59	680.00	43.50	10.12	219.57
T cells_abs [x10⁶]	377.00	68.00	1.74	3.39	60.60
T cells_(CD3+CD4+) [% of CD3⁺]	53.00	50.82	26.20	49.74	51.01
T cells_(CD3+CD8+) [% of CD3⁺]	47.02	49.20	73.85	50.31	49.01
IFN-γ ⁺ T cells [% of CD3 ⁺ ]	0.03	1.02	81.17	27.13	0.63
IFN-γ⁺ T cells [/μl]	0.85	6.94	35.50	2.74	1.38
IFN-γ ⁺ T cells [x10 ⁶ ]	0.11	0.69	1.42	0.92	0.38
IFN-γ⁻ T cells [% of CD3⁺]	99.97	98.98	18.83	72.87	99.37
IFN-γ⁻ T cells [/μl]	2834.59	673.00	8.20	7.37	218.12
IFN-γ⁻ T cells [x10⁶]	377.00	67.30	0.33	2.47	60.20
IFN-γ⁺ T cells_(CD3+) [% of CD4]	0.02	0.29	12.91	6.36	0.17
IFN-γ ⁺ T cells _(CD3+CD4+) [% of CD4]	0.04	0.58	50.63	13.28	0.41
IFN-γ⁺ T cells_(CD3+CD4+) [/μl]	0.60	2.00	5.78	0.67	0.46
IFN-γ ⁺ T cells _{abs (CD3+CD4+)} [x10 ⁶ ]	0.08	0.20	0.23	0.22	0.13
IFN-γ⁺ T cells_(CD3+) [% of CD8]	0.01	0.71	67.96	20.49	0.40
IFN-γ ⁺ T cells _(CD3+CD8+) [% of CD8]	0.02	1.43	91.84	40.21	0.73
IFN-γ⁺ T cells_(CD3+CD8+) [/μl]	0.27	4.79	29.50	2.04	0.79
IFN-γ ⁺ T cells _{abs (CD3+CD8+)} [x10 ⁶ ]	0.04	0.48	1.18	0.69	0.22
B	*2. Validation run*
	Leukapheresis	OF	TCF	WF	NF
volume [ml]	114	100	40	337	288
viability [%]	99.31	98.07	62.05	99.27	98.83
WBCs_(CD45+) [x10⁶/ml]	18.77	2.36	0.03	0.00	0.66
WBCs_abs [x10⁶]	2133.91	236.00	1.06	0.69	190.94
T cells_(CD3+CD56-) [% of WBCs]	16.97	36.69	27.92	25.73	41.15
T cells [/μl]	3175.44	865.00	7.43	0.53	272.92
T cells_abs [x10⁶]	362.00	86.50	0.30	0.18	78.60
T cells_(CD3+CD4+) [% of CD3⁺]	71.57	62.28	78.45	69.19	62.36
T cells_(CD3+CD8+) [% of CD3⁺]	28.50	37.75	21.80	30.88	37.71
IFN-γ ⁺ T cells [% of CD3 ⁺ ]	0.11	0.07	19.18	3.80	0.01
IFN-γ⁺ T cells [/μl]	3.50	0.61	1.42	0.02	0.03
IFN-γ ⁺ T cells [x10 ⁶ ]	0.40	0.06	0.05	0.01	0.01
IFN-γ⁻ T cells [% of CD3⁺]	99.89	99.93	80.82	96.20	99.99
IFN-γ⁻ T cells [/μl]	3175.44	864.00	6.00	0.51	272.92
IFN-γ⁻ T cells [x10⁶]	362.00	86.40	0.23	0.17	78.60
IFN-γ⁺ T cells_(CD3+) [% of CD4]	0.07	0.02	4.95	1.51	0.01
IFN-γ ⁺ T cells _(CD3+CD4+) [% of CD4]	0.10	0.03	6.43	1.93	0.02
IFN-γ⁺ T cells_(CD3+CD4+) [/μl]	2.27	0.16	0.38	0.01	0.03
IFN-γ ⁺ T cells _{abs (CD3+CD4+)} [x10 ⁶ ]	0.26	0.02	0.02	0.00	0.01
IFN-γ⁺ T cells_(CD3+) [% of CD8]	0.04	0.04	13.84	2.18	0.00
IFN-γ ⁺ T cells _(CD3+CD8+) [% of CD8]	0.12	0.10	62.96	6.52	0.00
IFN-γ⁺ T cells_(CD3+CD8+) [/μl]	1.09	0.32	1.02	0.01	0.00
IFN-γ ⁺ T cells _{abs (CD3+CD8+)} [x10 ⁶ ]	0.12	0.03	0.04	0.00	0.00
C	*3. Validation run*
	Leukapheresis	OF	TCF	WF	NF
volume [ml]	109	100	43	330	263
viability [%]	99.56	97.86	58.92	90.43	98.21
WBCs_(CD45+) [x10⁶/ml]	15.73	1.62	0.12	0.01	0.53
WBCs_abs [x10⁶]	1714.31	162.00	5.01	4.82	138.49
T cells_(CD3+CD56-) [% of WBCs]	12.76	30.94	36.52	31.07	28.56
T cells [/μl]	2009.17	500.00	42.56	4.55	150.19
T cells_abs [x10⁶]	219.00	50.00	1.83	1.50	39.50
T cells_(CD3+CD4+) [% of CD3⁺]	75.96	72.60	87.29	80.31	70.89
T cells_(CD3+CD8+) [% of CD3⁺]	24.08	27.43	12.81	19.71	29.13
IFN-γ ⁺ T cells [% of CD3 ⁺ ]	0.06	1.11	63.13	36.29	0.47
IFN-γ⁺ T cells [/μl]	1.20	5.55	26.74	1.65	0.71
IFN-γ ⁺ T cells [x10 ⁶ ]	0.13	0.56	1.15	0.54	0.19
IFN-γ⁻ T cells [% of CD3⁺]	99.94	98.89	36.87	63.71	99.53
IFN-γ⁻ T cells [/μl]	2009.17	494.00	15.67	2.89	149.81
IFN-γ⁻ T cells [x10⁶]	219.00	49.40	0.67	0.95	39.40
IFN-γ⁺ T cells_(CD3+) [% of CD4]	0.06	0.87	53.17	30.37	0.47
IFN-γ ⁺ T cells _(CD3+CD4) [% of CD4]	0.06	1.10	58.25	34.13	0.49
IFN-γ⁺ T cells_(CD3+CD4+) [/μl]	0.91	3.99	21.63	1.24	0.52
IFN-γ ⁺ T cells _{abs (CD3+CD4+)} [x10 ⁶ ]	0.10	0.40	0.93	0.41	0.14
IFN-γ⁺ T cells_(CD3+) [% of CD8]	0.02	0.30	11.48	7.75	0.10
IFN-γ ⁺ T cells _(CD3+CD8+) [% of CD8]	0.00	0.99	88.29	37.27	0.30
IFN-γ⁺ T cells_(CD3+CD8+) [/μl]	0.00	1.36	4.81	0.33	0.13
IFN-γ ⁺ T cells _{abs (CD3+CD8+)} *[10** ⁶ ]	0.00	0.14	0.21	0.11	0.03

The purification of clinical-grade CMVpp65-specific CD4+ and CD8+ T cells from three healthy CMV-seropositive donors was performed aseptically under GMP conditions using the IFN-γ-based CliniMACS CCS system and the GMP-compliant CMVpp65pp for short term ex vivo stimulation. Detailed information for all three validation processes (A-B) including viability [%], viable cell number [x106/ml; x106; /μl] and specific cell frequencies [%] for all CliniMACS CCS fractions were determined. The results for the representative analysis of the cells from the leukapheresis, original fraction (OF), T-cell fraction (TCF), negative fraction (NF), and waste fraction (WF) are shown. Bold data reflected the results obtained for the CMV-specific IFN-γ-positive T cells.

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ISSN: 1479-5876

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