Immunotoxicity profile of natalizumab

Figures & data

Table 1.  Summaries of the studies.

Study no.	Study type	Species	Duration	Natalizumab dose levels	Dose frequency	Immunotoxicity endpoints and measurements
1	Immunotoxicity – in vitro	Human PBMC and monocytes	NA	10 ng/ml – 100 μg/ml	NA	Proliferation capacity Cytokine production Cytotoxic T-cell activity Natural killer activity
2	Toxicity – in vivo and ex vivo	Cynomolgus monkey	4 weeks	0.3, 3, 30 mg/kg	Every other day	Hematology Immunophenotyping Proliferation capacity (PBMC) Natural killer activity (PBMC) Immune organ weights and histopathology
3	Toxicity – in vivo	Cynomolgus monkey	6 months	3, 10, 30, 60 mg/kg	Weekly	Hematology Immunophenotyping Immune organ weights and histopathology
4	Toxicity – in vivo	Cynomolgus monkey – juvenile	6 months	10, 30, 60 mg/kg	Weekly	Hematology Immunophenotyping Immune organ weights and histopathology
5	Immunotoxicity – in vivo	Cynomolgus monkey	6 weeks	3, 30 mg/kg	Weekly	Hematology TDAR – Anti-KLH and Anti-TT Antibody
6	Toxicity – in vivo	Rhesus monkey	4 weeks	30, 60 mg/kg	Weekly	Hematology Immunophenotyping Immune organ weights and histopathology Immune organ immuno- histochemistry

NA, not applicable; PBMC, peripheral blood mononuclear cells; TDAR, T-cell-dependent antibody response; KLH, keyhole limpet hemocyanin; TT, tetanus toxoid.

Table 2.  Natalizumab exposure levels.

Study	Study duration	Natalizumab dose (mg/kg)	Parameter^e	Natalizumab pre-dose Day 8 (μg/ml)	Natalizumab post-dose Day 8 (μg/ml)	Natalizumab pre-last dose (μg/ml)	Natalizumab post-last dose (μg/ml)	Natalizumab antibody pre-last dose (μg/ml)
2^a	28 days	0.3	Mean ± SD		0.7 ± 0.4 (10/10)		0.7 (2/10)	31.9 ± 23.9 (10/10)
			Range		0.2–1.4		0.5–1.0	8.2–79.7
		3.0	Mean ± SD		81.0 ± 28.5 (10/10)		15.4 ± 30.3 (4/10)	25.3 ± 33.8 (9/10)
			Range		16.7–121.0		0.1–60.8	10.0–115.0
		30.0	Mean ± SD		1786.0 ± 2126.0 (10/10)		1173.0 ± 1006.0 (10/10)	15.6 (1/10)
			Range		887.0–7820.0		291.0–3430.0	NA
3^b	6 months	3	Mean ± SD	1.2 ± 0.4 (6/6)	58.1 ± 10.1 (6/6)	0 (0/6)	0.4 ± 0.1 (3/6)	477.5 ± 298.7 (6/6)
			Range	0.8–1.8	47.9–69.0	NA	0.3 -0.5	272.2–1057.7
		10	Mean ± SD	9.4 ± 3.4 (5/6)	272.5 ± 41.5 (6/6)	0 (0/5)	21.3 ± 15.4 (4/5)	1332 ± 1674 (5/5)
			Range	7.1–15.4	229.1–336.9	NA	1.1–38.2	370.7–4296.5
		30	Mean ± SD	63.7 ± 62.4 (10/10)	706.5 ± 120.4 (10/10)	350.5 ± 312.3 (5/10)	737.5 ± 620.2 (10/10)	1085.0 ± 674.8 (6/10)
			Range	24.1–181.6	553.5–896.3	11.8–805.4	161.6–1632.0	563.9–2337.6
		60	Mean ± SD	282.1 ± 138.4 (10/10)	1734.6 ± 150.1 (10/10)	1668.7 ± 1321.0 (6/10)	2313.1 ± 1450.3 (10/10)	391.1 ± 250.5 (4/10)
			Range	72.8–471.2	1559.2–2084.6	153.3–3308.6	905.8–5558.8	24.6–560.6
4^b	6 months	10	Mean ± SD	19.4 ± 5.1 (6/6)	236.2 ± 18.0 (6/6)	54.0 (1/6)	131.5 ± 162.9 (3/6)	1879.4 ± 2027.2 (5/6)
			Range	14.4–27.9	213.4–267.0	NA	1.0–314.0	169.1–5162.4
		30	Mean ± SD	115.1 ± 41.0 (6/6)	767.4 ± 176.2 (6/6)	431.8 ± 266.5 (3/6)	663.5 ± 544.0 (5/6)	1624.7 ± 1218.4 (3/6)
			Range	34.3–144.6	619.5–1073.9	124.8–603.6	131.4–1278.0	585.7–2965.6
		60	Mean ± SD	284.0 ± 131.0 (10/10)	1462.6 ± 353.7 (10/10)	1255.1 ± 663.8 (9/9)	2261.3 ± 617.0 (9/9)	0 (0/9)
			Range	138.9–564.2	756.1–1931.0	460.8–2801.7	1464.0–3586.9	NA
5^c	6 weeks	3	Mean ± SD	12.0 ± 4.0 (5/5)	78.0 ± 15.0 (5/5)	0 (0/6)	1.0 ± 0.4 (5/6)	584 ± 220 (6/6)
			Range	6.0–18.0	55.0–89.0	NA	0.4–1.0	467.0–1030.0
		30	Mean ± SD	257.0 ± 130.0 (6/6)	1233.0 ± 151.0 (6/6)	316 ± 292 (4/6)	692.0 ± 595.0 (6/6)	451 ± 400 (3/6)
			Range	130.0–380.0	1100.0–1500.0	3.8–580.0	21.0–1400.0	48.7–849.0
6^d	22 days	30	Mean ± SD	158.2 ± 65.2 (10/10)	705.4 ± 96.1 (10/10)	109.7 ± 134.4 (5/10)	467.9 ± 203.8 (10/10)	13.0 (1/10)
			Range	51.9–244.1	592.5–840.9	3.6–298.3	284.8–825.4	NA
		60	Mean ± SD	333.2 ± 123.7 (10/10)	1500.9 ± 184.1 (10/10)	379.0 ± 371.1 (7/10)	1357.7 ± 431.5 (10/10)	9.7 (1/10)
			Range	127.1–507.1	1351.8–1774.2	24.7–1155.1	757.1–2197.6	NA

BLQ, below limit of quantification; NA, not applicable.

Note: With the exception of a single animal in the 3 mg/kg group of Study 2 with an anti-natalizumab antibody concentrations of 9.9 μg/ml, all animals were negative for antibody at the Day 8 pre-dose time point.

^aNatalizumab and antibody concentration measured 24 hours after previous dose; data for Days 8 and 28; every other day dosing starting Day 1.

^bNatalizumab concentration measured before dosing and 30 minutes post-dosing and antibody concentration measured before dosing; data for Days 8 and 176; weekly dosing starting Day 1.

^cNatalizumab concentration measured before dosing and 3 hour post-dosing and antibody concentration measured before dosing; data for Days 8 and 36; weekly dosing starting Day 1.

^dNatalizumab concentration measured before dosing and 30 minutes post-dosing and antibody concentration measured before dosing; data for Days 8 and 22; weekly dosing starting Day 1.

^eMean value ± SD (X/X) and Range for all animals positive for analyte. Numbers in parentheses indicate the number of animals positive for the analyte/Number of animals total.

Figure 1.  (A) White blood cell and (B) lymphocyte counts in Study 3.

Table 3.  Mean change in white blood cell (WBC) and lymphocyte counts in studies of cynomolgus and rhesus monkeys receiving natalizumab.

Study	Natalizumab dose (mg/kg)	Study day	Fold change in cell counts vs. control animals
			WBC	Lymphocytes
2	0.3	8	1.31	1.71
	3		1.65	2.26
	30		1.48	2.28
3	3	22	0.98	1.31
	10		1.06	1.48
	30		1.42	2.18
	60		1.58	2.43
4	10	23	1.37	1.63
	30		1.90	2.34
	60		1.82	2.28
5	3	28	1.04	0.83
	30		1.78	1.99
6	30	2	1.32	1.94
	60		1.41	2.35

Results from the first hematology samples evaluated during treatment in each study; representative of results seen for all evaluated times during treatment. For all studies, hematology sampling occurred 1 day after a given dose.

Figure 2.  Phenotype analysis of circulating leukocytes in Study 4 by (A) percentages and (B) absolute counts of cellular subsets.

Table 4.  Summary of phenotyping of WBC populations in monkeys treated with natalizumab.

Study no.	Species	Duration/Dose Frequency	Phenotyping Marker	Mean (±SD) % of Cells Positive at Natalizumab dose levels of:
				0	0.3	3	10	30	60
2	Cynomolgus monkey	4 weeks/Every other day	CD2	65.23 ± 9.04	65.91 ± 4.07	60.52 ± 6.90	NA	58.69 ± 6.14	NA
			CD4	29.83 ± 6.68	32.14 ± 5.06	28.31 ± 4.85		30.32 ± 4.75
			CD8	36.34 ± 6.16	36.44 ± 6.30	32.71 ± 4.49		29.03 ± 4.24
			CD14	1.71 ± 1.10	1.62 ± 1.46	2.91 ± 1.41*		2.69 ± 1.18*
			CD20	17.69 ± 7.58	19.46 ± 4.68	24.80 ± 6.05		27.07 ± 5.78
3	Cynomolgus monkey	6 months/ Weekly	CD34	≤ 0.01	NA	≤ 0.01	≤ 0.01	≤ 0.01	≤ 0.01
4	Cynomolgus monkey – juvenile	6 months/ Weekly	CD3	59.15 ± 11.48	NA	NA	53.56 ± 9.97	51.60 ± 9.47	49.54 ± 8.30
			CD4	33.91 ± 6.14			33.73 ± 7.49	29.09 ± 5.23	26.70 ± 5.52
			CD8	29.78 ± 10.29			23.12 ± 6.42	23.41 ± 6.33	22.71 ± 4.29
			CD20	23.08 ± 11.81			31.44 ± 11.08	37.42 ± 9.79	36.70 ± 6.45
			CD34	5.50 ± 1.75			5.41 ± 1.76	3.97 ± 1.13	2.77 ± 1.72
6	Rhesus monkey	4 weeks/ Weekly	CD3	62.07 ± 24.71	NA	NA	NA	57.42 ± 11.45	55.13 ± 9.79
			CD4	35.12 ± 14.82				32.26 ± 4.99	30.71 ± 4.6
			CD8	26.19 ± 7.63				24.49 ± 7.61	24.19 ± 8.62
			CD20	19.38 ± 10.97				20.99 ± 7.70	24.33 ± 6.79

*Statistically significant at p ≤ 0.05 for males only.

NA, not applicable.

Markers evaluated included CD2 (lymphocytes, monocytes, NK cells), CD3 (T-cells), CD4 (T-helper cells), CD8 (T-suppressor cells), CD14 (monocytes), CD20 (B-cells) and CD34 (immature hematopoietic cells). Percentage of cells was determined as the percent of all WBC for CD2, CD14, and CD34 and as the percent of all lymphocytes for CD3, CD4 and CD8.

Table 5.  Results of immune function tests in lymphocytes and spleen cells obtained from cynomolgus monkeys in Study 2 that were treated with natalizumab 30 mg/kg every other day for 4 weeks.

Natalizumab dose
	PBMC basal natural killer cell activity (% lysis)
	100:1 E:T ratio	33:1 E:T ratio	11:1 E:T ratio
0 mg/kg	30.24 ± 30.78	21.27 ± 23.96	12.42 ± 19.37
30 mg/kg	34.29 ± 19.81	22.07 ± 10.06	11.19 ± 4.19
	Spleen cell basal natural killer cell activity (% lysis)
	100:1 E:T ratio	33:1 E:T ratio	11:1 E:T ratio
0 mg/kg	35.07 ± 11.60	16.42 ± 7.88	6.79 ± 4.13
30 mg/kg	28.64 ± 14.23	15.89 ± 12.06	7.88 ± 9.34
	PHA-stimulated proliferation of PBMC (OD at 450 nm)
	1 μg/ml	2.5 μg/ml	5 μg/ml
0 mg/kg	0.083 ± 0.110	0.159 ± 0.157	0.246 ± 0.203
30 mg/kg	0.269 ± 0.169	0.339 ± 0.171	0.378 ± 0.139
	PHA-stimulated proliferation of spleen cells (OD at 450 nm)
	1 μg/ml	2.5 μg/ml	5 μg/ml
0 mg/kg	0.048 ± 0.057	0.101 ± 0.124	0.147 ± 0.140
30 mg/kg	0.192 ± 0.122	0.253 ± 0.126	0.289 ± 0.139
	LPS-stimulated proliferation of PBMC (OD at 450 nm)
	5 μg/ml	20 μg/ml	50 μg/ml
0 mg/kg	0.012 ± 0.024	0.025 ± 0.39	0.040 ± 0.084
30 mg/kg	0.030 ± 0.040	0.021 ± 0.024	0.035 ± 0.039
	LPS-stimulated proliferation of spleen cells (OD at 450 nm)
	5 μg/ml	20 μg/ml	50 μg/ml
0 mg/kg	0.053 ± 0.074	0.059 ± 0.066	0.057 ± 0.060
30 mg/kg	0.051 ± 0.045	0.069 ± 0.057	0.092 ± 0.086

PBMC, peripheral blood mononuclear cells; OD, optical density; PHA, phytohemag-glutinin; LPS, lipopolysaccharide; E:T ratio, effector:target ratio.

Figure 3.  (A) IgM and (B) IgG responses to immunization with keyhole limpet hemocyanin (KLH; Days 7, 35, and 63) and tetanus toxoid (TT; Days 56 and 70) in Study 5. Closed symbols/solid lines, anti-KLH antibodies (center-point titer); open symbols/dotted lines, anti-TT antibodies (cut-point titer); circles, control; triangles, natalizumab 3 mg/kg; squares, natalizumab 30 mg/kg; *p < 0.05 vs. control.

Table 6.  Anti-KLH and anti-TT titers in monkeys treated with natalizumab.

	Group 1			Group 2			Group 3
	Vehicle			3 mg/kg Natalizumab			30 mg/kg Natalizumab
	Responders^a	IgM Titer^b	IgG Titer^b	Responders^a	IgM Titer^b	IgG Titer^b	Responders^a	IgM Titer^b	IgG Titer^b
Anti-KLH Titers
Day 17	6/6, 6/6	515 ± 222	1004 ± 215	6/6, 6/6	371 ± 76	988 ± 466	6/6, 6/6	302 ± 42*	834 ± 250
Day 21	6/6, 6/6	461 ± 134	1189 ± 325	6/6, 6/6	366 ± 64	1238 ± 638	6/6, 6/6	311 ± 40*	968 ± 355
Day 28	6/6, 6/6	489 ± 258	1467 ± 524	6/6, 6/6	368 ± 123	1539 ± 806	6/6, 6/6	276 ± 70	1188 ± 510
Day 35 Pre	6/6, 6/6	427 ± 215	1689 ± 651	5/6, 6/6	309 ± 146	1801 ± 1053	6/6, 6/6	312 ± 90	1656 ± 987
Day 45	5/6, 6/6	754 ± 857	4232 ± 1864	6/6, 6/6	499 ± 159	4405 ± 3588	6/6, 6/6	514 ± 151	4168 ± 2670
Day 49	5/6, 6/6	670 ± 723	4340 ± 2019	6/6, 6/6	497 ± 129	4793 ± 3706	6/6, 6/6	488 ± 147	4220 ± 2617
Day 56	5/6, 6/6	512 ± 453	4219 ± 2225	5/6, 6/6	564 ± 404	5184 ± 4142	5/6, 6/6	377 ± 184	3861 ± 2513
Day 63 Pre	5/6, 6/6	400 ± 303	3890 ± 2446	5/6, 6/6	417 ± 252	4425 ± 3782	5/6, 6/6	300 ± 178	4023 ± 2939
Day 73	5/6, 6/6	498 ± 395	5765 ± 2682	5/6, 6/6	554 ± 292	6055 ± 4178	5/6, 6/6	354 ± 214	6310 ± 4171
Day 77	5/6, 6/6	612 ± 649	6038 ± 2637	5/6, 6/6	520 ± 259	6625 ± 4668	5/6, 6/6	339 ± 167	6379 ± 4141
Day 84	5/6, 6/6	579 ± 730	5756 ± 2862	5/6, 6/6	511 ± 231	6531 ± 4688	5/6, 6/6	344 ± 184	5962 ± 3967
Day 91	5/6, 6/6	502 ± 547	4939 ± 2596	5/6, 6/6	473 ± 330	5396 ± 4042	5/6, 6/6	353 ± 179	5161 ± 3312
Anti-TT Titers
Day 56 Pre	0/6, 0/6	N/A	N/A	0/6, 1/6	N/A	N/A	0/6, 1/6	N/A	N/A
Day 66	5/6, 1/6	450 ± 163	50	6/6, 0/6	383 ± 207	N/A	3/6, 0/6	442 ± 170	N/A
Day 70 Pre	6/6, 4/6	2142 ± 1410	138 ± 118	6/6, 5/6	1488 ± 858	120 ± 110	5/6, 1/6	1550 ± 716	N/A
Day 80	6/6, 6/6	1467 ± 1055	1883 ± 904	5/6, 6/6	1320 ± 607	1267 ± 728	5/6, 6/6	840 ± 275	1692 ± 2221
Day 84	6/6, 6/6	1300 ± 875	1642 ± 671	5/6, 6/6	1150 ± 652	1133 ± 516	5/6, 6/6	760 ± 227	1458 ± 1288
Day 91	6/6, 6/6	758 ± 645	1417 ± 571	4/6, 6/6	1313 ± 554	1217 ± 725	5/6, 6/6	600 ± 165	1192 ± 1191

^aNumber of animals with a positive antibody response/number of animals in group; (IgM responders, IgG responders)

^bMean ± SD

Dosing: Days 1, 8, 15, 22, 29, 36

KLH immunization: Days 7, 35, 63

TT immunization: Days 56, 70

Pre = Sample taken prior to test article administration and/or immunization procedures scheduled for that day

N/A = Not applicable as no IgM or IgG response was detected in any animal.

Table 7.  Results of in vitro immune function tests in PBMC and monocytes from healthy human volunteers (N=5).

Natalizumab concentration	In vitro immune function test
	AntiCD3 stimulated proliferation of PBMC (OD at 450 nm)
	0 ng/ml AntiCD3	10 ng/ml AntiCD3		100 ng/ml AntiCD3	1000 ng/ml AntiCD3
0 μg/ml	0.814 ± 0.084	1.174 ± 0.127		1.022 ± 0.132	0.999 ± 0.122
100 μg/ml	0.945 ± 0.098	1.249 ± 0.121		1.140 ± 0.126	1.156 ± 0.106
	PHA-stimulated proliferation of PBMC (OD at 450 nm)
	0 μg/ml PHA	0.5 μg/ml PHA		1 μg/ml PHA	5 μg/ml PHA
0 μg/ml	0.795 ± 0.094	0.849 ± 0.026		1.033 ± 0.043	1.352 ± 0.098
100 μg/ml	0.988 ± 0.104	0.984 ± 0.026*		1.159 ± 0.066	1.331 ± 0.097
	Anti-CD3-mediated cytokine production by PBMC (pg/ml)
	IL1β	IL2	IL6	IL-10	TNFβ
0 μg/ml	574.66 ± 117.76	421.70 ± 112.23	117.35 ± 57.00	680.50 ± 118.18	468.84 ± 89.63
100 μg/ml	562.34 ± 122.42	481.08 ± 113.29	216.80 ± 38.61	627.48 ± 129.80	491.02 ± 90.83
	LPS-mediated cytokine production by monocytes (pg/ml)
	IL1α		TNFα
0 μg/ml	381.08 ± 16.50		1061.94 ± 89.94
100 μg/ml	376.52 ± 28.73		1060.92 ± 58.45
	Basal natural killer cell activity (% Lysis)
	100:1 E:T ratio	33:1 E:T ratio		11:1 E:T ratio
0 μg/ml	59.56 ± 8.09	37.15 ± 11.18		15.84 ± 6.32
100 μg/ml	41.90 ± 6.37	35.47 ± 14.21		20.85 ± 14.60
	IL2-augmented natural killer cell activity (% lysis)
	100:1 E:T ratio	33:1 E:T ratio		11:1 E:T ratio
0 μg/ml	77.66 ± 6.87	56.86 ± 10.14		28.97 ± 8.64
100 μg/ml	59.13 ± 4.45	48.73 ± 8.57		27.26 ± 11.65
	Anti-CD3-mediated T-cell cellular cytotoxicity (% lysis)
	100:1 E:T ratio	50:1 E:T ratio		25:1 E:T ratio
0 μg/ml	43.59 ± 6.98	41.68 ± 4.63		26.43 ± 3.11
100 μg/ml	42.21 ± 10.18	32.20 ± 4.94		18.49 ± 3.70

*p ≤ 0.05 vs. control.

PBMC, peripheral blood mononuclear cell; PHA, phytohemagglutinin; OD, optical density; IL, interleukin; TNF, tumor necrosis factor; E:T ratio, effector:target ratio.