Characterization and non-clinical assessment of the proposed etanercept biosimilar GP2015 with originator etanercept (Enbrel^®)

Figures & data

Figure 1. Panel of assays to measure (in vitro) ligand binding and effector function of Enbrel /GP2015 in relation to the structure of the etanercept molecule.

Table 1. The experimental groups in the human TNF transgenic mouse model Tg197 [24] used to compare the therapeutic efficacy of GP2015 and Enbrel at a pharmacologically sensitive dose of 10 mg/kg.

Group no	Test article	Dose (mg/kg)	Dose frequency*	Dose volume (ml/kg)	Route of adm.	Animal number	Age at sacrifice
1	Enbrel	10 mg/kg	Single	10	IP	10m/10f	6 weeks
2	GP201S	10 mg/kg	Single	10	IP	10m/10f	6 weeks
3	Enbrel	10 mg/kg	2/week	10	IP	10m/10f	8 weeks
4	GP201S	10 mg/kg	2/week	10	IP	10m/10f	8 weeks
5	Enbrel	10 mg/kg	2/week	10	IP	10m/10f	10 weeks
6	GP201S	10 mg/kg	2/week	10	IP	10m/10f	10 weeks
7	Vehicle control	0 mg/kg	2/week	10	IP	Sm/Sf	10 weeks
8	Enbrel positive control	30 mg/kg	2/week	10	IP	Sm/Sf	10 weeks
6 week Cons	None	None	None	–	–	2m/2f	6 weeks

*Start of administration at age of 6 weeks.

Table 2. GP2015/Enbrel neutralization of TNF-α and LT-α in an RGA, binding to TNF-α and Fc receptors (SPR; Biacore assay).

	Enbrel				GP2015
Assay	Range	Mean	Median	SD	Range	Mean	Median	SD
Functional bioassays: neutralization and binding
TNF-α neutralization (luciferase RGA)^a	76–118	89	87	8	93–101	97	97	2
TNF-α neutralization (apoptosis)^a	92–134	115	115	12	92–109	101	102	6
LT-α neutralization (luciferase RGA)^a	78–123	94	95	2	90–96	94	94	2
TNF-α binding (SPR)^a	85–101	96	95	4	89–101	96	97	4
Binding to human C1q (ELISA)^a	113–118	115	115	2	110–115	113	113	2
Functional bioassays: cytotoxicity activity (CDC and ADCC)
CDC^a	59–101	78	79	10	93–132	106	103	12
ADCC^a	196–217	204	199	9	79–121	92	91	13
Fc receptor panel binding
Low-affinity Fc receptors (KD in µM)
FcγRIIa	10.1–14.4	13.0	13.8	1.8	11.6–13.8	13.0	13.3	0.8
FcγRIIb	25.8–36.2	31.3	30.5	4.0	29.1–37.5	33.1	32.4	2.6
FcγRIIIa F158	17.2–21.0	18.8	18.8	1.4	19.3–29.8	24.5	24.1	3.7
FcγRIIIa V158	7.9–10.6	8.9	8.5	1.0	9.2–13.5	11.6	11.8	1.7
FcγRIIIb	20.8–29.4	26.0	26.3	3.3	20.4–35.7	27.4	26.9	4.8
High-affinity Fc receptors (KD)
FcγRIa [nM]	34.7–41.7	36.5	35.3	2.7	32.9–57.3	39.7	37.6	7.5
FcRn [mM]	130–161	147	150	12	141–165	153	154	8

The range of results from analysis of different batches is given.

^aFinal results are expressed as relative potency (%) of a sample compared with a reference standard. Ranges comprise data for both drug substance and drug product.

TNF: Tumor necrosis factor; SPR: surface plasmon resonance; RGA: reporter-gene assay; ADCC: antibody-dependent cellular cytotoxicity; CDC: complement-dependent cytotoxicity; K_D: equilibrium dissociation constant; SD: standard deviation.

Table 3. Ratios of mean PK parameters in male rabbits for five GP2015 formulations relative to Enbrel/EU [25 mM phosphate/25 mM arginine HCl buffer].

Test article	Buffer formulation		Cmax	tmax	t1/2	Kel	AUC0–168 h	AUC0–∞
GP2015 originator^a	25 mM phosphate	25 mM arginine HCl	1.20	0.98	1.08	0.93	1.23	1.23
GP2015	50 mM citrate	25 mM lysine	1.19	1.07	0.95	1.05	1.28	1.28
GP2015	25 mM citrate	25 mM lysine	1.44	0.83	0.91	1.10	1.35	1.35
GP2015	25 mM citrate	25 mM proline	2.02	0.61	1.04	0.95	1.52	1.52
GP2015	25 mM phosphate	25 mM lysine	1.55	0.64	0.98	1.00	1.37	1.37

^aThe originator-like formulation has an identical buffer composition as Enbrel/US.

AUC_0–168 h: Area under the serum concentration–time curve from time zero to time 168 h; AUC_0–∞: area under the serum concentration–time curve from time zero to infinity; C_max: maximum observed serum concentration; K_el: elimination rate constant; t_max: time to the maximum observed serum concentration; t_1/2: elimination half-life.

Figure 2. Dose-dependent inhibition of TNF-α function by GP2015 and Enbrel in a cellular NFκB-dependent luciferase reporter gene assay.

Figure 3. Results for TNF-α neutralization for different batches of Enbrel (Originator) and GP2015 (Biosimilar) in the reporter-gene assay.

Figure 4. Comparative pharmacokinetic profiles after single 8 mg/kg bodyweight (b.w.) subcutaneous (s.c.) administration to rabbits.

Figure 5. Arthritic score assessment in the human TNF transgenic mouse model Tg197 [25]. At a pharmacologically sensitive dose of 10 mg/kg, GP2015 and Enbrel were indistinguishable in inhibiting arthritic disease symptoms when compared to vehicle buffer-treated control group.

Figure 6. The histopathology and in-life mean arthritic score at week 10 in experimental Tg197 mice. By week 10 the mean disease severity scores of the biweekly treated groups (from week 6 onwards), were as follows: (G5) Enbrel at 10 mg/kg = 3.13 (HS) and 1.04 (AS), (G6) GP2015 at 10 mg/kg = 2.89 (HS) and 0.9 (AS), (G7) vehicle GP2015 buffer = 3.58 (HS) and 1.43 (AS), (G8) Enbrel at 30 mg/kg = 1.53 (HS) and 0.61 (AS) Control mice at week 6 had a score of 2.81 (HS) and 1.19 (AS). Bars indicate standard errors of the mean.

Table 4. Mean ± standard deviation toxicokinetic parameters following subcutaneous administration of GP2015 or Enbrel 15 mg/kg subcutaneously (once every 3 days for 4 weeks) to groups of cynomolgus monkeys (n = 3 in each group).

Gender [day]	Treatment group	Cmax (µg/ml)	tmax (h)	AUC0–T (µg/ml h)	t1/2 (h)
Male [1]	GP2015	78.4 ± 9.9	24.7 ± 7.0	4020 ± 164	45.9 ± NC
Female [1]	GP2015	83.9 ± 22.3	22.0 ± 3.5	4390 ± 1050	59.4 ± 16.3
Male [1]	Enbrel	77.9 ± 24.0	24.0 ± 0.0	4050 ± 463	61.1 ± NC
Female [1]	Enbrel	62.2 ± 5.8	29.3 ± 4.6	3230 ± 275	50.4 ± NC
Male [7]	GP2015	98.9 ± 8.2	18.0 ± 10.4	5570 ± 690	55.8 ± NC
Female [7]	GP2015	95.9 ± 13.5	22.7 ± 10.1	5610 ± 223	66.1 ± NC
Male [7]	Enbrel	122.0 ± 33.6	16.0 ± 9.2	6300 ± 810	62.0 ± 29.3
Female [7]	Enbrel	109.0 ± 6.4	24.0 ± 0.0	6200 ± 71.2	83.2 ± 26.6
Male [28]	GP2015	49.3 ± 39.7	22.0 ± 3.5	2180 ± 1950	13.8 ± 2.8
Female [28]	GP2015	27.1 ± 14.6	24.0 ± 0.0	987 ± 653	7.9 ± 2.7
Male [28]	Enbrel	38.7 ± 55.8	20.0 ± 6.9	2000 ± 3100	12.6 ± 8.2
Female [28]	Enbrel	34.9 ± 22.0	26.7 ± 4.6	1500 ± 1240	13.2 ± 6.8

C_max: Maximum serum concentration; t_max: time to reach C_max; AUC_0–T: area under the serum concentration–time curve; t_1/2: elimination half-life; NC: not calculable.

Campa M, Ryan C, Menter A. An overview of developing TNF-α targeted therapy for the treatment of psoriasis. Expert Opin Investig Drugs. 2015;24:1343–1354. doi:10.1517/13543784.2015.1076793.

 PubMed Web of Science ®Google Scholar

Keffer J, Probert L, Cazlaris H, et al. Transgenic mice expressing human tumour necrosis factor: a predictive genetic model of arthritis. EMBO J. 1991;10:4025–4031.

 PubMed Web of Science ®Google Scholar

Alten R, Cronstein BN. Clinical trial development for biosimilars. Semin Arthritis Rheum. 2015;44:S2–S8. doi:10.1016/j.semarthrit.2015.04.002.

 PubMed Web of Science ®Google Scholar

Kaymakcalan Z. Beam C and Salfeld J. Murine model for assessing adalimumab, infliximab and etanercept to prevent polyarthritis. Ann Rheum Dis. 2003;62(Suppl 1):136–137.

 Google Scholar

Enbrel BLA 98-0286. [updated 1998 Nov 2; cited 2015 Mar 17]. Available from: http://www.accessdata.fda.gov/scripts/cder/drugsatfda/index.cfm/.

 Google Scholar