In vitro human helper T-cell assay to screen antibody drug candidates for immunogenicity

Figures & data

Figure 1. Gating strategy of flow cytometry for BrdU⁺ T_H cells in PBMC stained with antibodies against CD3, CD4, and BrdU. (A–C) Representative results for cells with nontreatment control. (D–F) Representative results for cells treated with hA33. (A and D) PBMC gated for CD4⁺CD14⁻ cells. (B and E) Cells from A and D gated for CD3⁺. (C and F) Cells from B and E gated for BrdU⁺. (F) Final selected population is BrdU⁺CD3⁺CD4⁺CD14⁻ that correspond to T_H cells that might proliferate in the assays.

Figure 2. Time dependency for proliferation of T_H cells among PBMC treated with KLH (n = 81 donor samples total tested).

Figure 3. Frequency distribution of Stimulation Index (SI) in response to treatment with etanercept. (n = 218 donor samples total tested).

Figure 4. Stimulation index (SI) distribution in donor samples treated with KLH (n = 81 donor samples total tested).

Figure 5. Stimulation index (SI) distribution among PBMC treated with therapeutic mAb. There were n = 218, 20, 54, 54, 22, 256 donor samples total treated with Etanercept, Basiliximab, Infliximab, Adalimumab, Abcicimab, and hA33, respectively.

Table 1. Estimated sample size for statistical significance of positive response.

Statistical Power: 80%
Antibody	Infliximab	Adalimumab	Abciximab	hA33
n	104	39	36	19
Statistical Power: 90%
Antibody	Infliximab	Adalimumab	Abciximab	hA33
n	136	49	45	25

Table 2. Frequency of positive samples for each mAb in the T_H cell assay.

Antibody	Etanercept	Basiliximab	Infliximab	Adalimumab	Abciximab	hA33
Total number donors	218	20	54	54	22	256
Number of high-response donors	8	0	8	14	6	101
Frequency of positive donors	3.7%	0.0%	14.8%	25.9%	27.3%	39.5%

Table 3. Clinical ADA incidence of mAb drugs.

Antibody	Target	ADA incidence		Donors	References
		%	n
Etanercept	TNFα	0.0	36	Healthy volunteer	a
		9.8	2989	RA*, AS*, Psoriasis	b
Infliximab	TNFα	50.0	32	Healthy volunteer	c
		30.8	2719	RA*, CD*, AS*, Psoriasis	d
Adalimumab	TNFα	29.4	75	Healthy volunteer	e
		15.0	1795	RA*, CD*, AS*, Psoriasis	f
Basiliximab	IL-2R	2.0	511	Transplant rejection	g
Abciximab	GPIIb/IIIa	17.8	1729	Coronary angioplasty	h
hA33	A33	62.2	53	Colon cancer	i

*RA: Rheumatoid arthritis; CD: Crohn’s disease; AS: Ankylosing spondylitis

^aSchultz et al. 2017.

^bMahil et al. 2013; Papp et al. 2005; Gordon et al. 2006; Leonardi et al. 2010; Tyring et al. 2007.

^cKubo et al. 2010.

^dMaini et al. 1998; Hanauer et al. 2002; Baert et al. 2003; Gottlieb et al. 2004, 2012; Sands et al. 2004; Reich et al. 2005; Bendtzen et al. 2006; Krathen et al. 2006; Wolbink et al. 2006; Menter et al. 2007; Vermeire et al. 2007; de Vries et al. 2009; Adişen et al. 2010; Torii et al. 2010, 2011; Hoffmann et al. 2011; Takahashi et al. 2013; Mazilu et al. 2014; Zisapel et al. 2015.

^ePharmaceuticals and Medical Devices Agency 2005.

^fBartelds et al. 2007; Sandborn et al. 2007; Menter et al. 2008; West et al. 2008; de Vries et al. 2009; Asahina et al. 2010; Lecluse et al. 2010; van Kuijk et al. 2010; Krieckaert et al. 2012; Mahil et al. 2013; Takahashi et al. 2013; Paramarta and Baeten 2014; Zisapel et al. 2015.

^gUS Food and Drug Administration 2001.

^hTcheng et al. 2001; Dery et al. 2004; US Food and Drug Administration 2013.

ⁱRitter et al. 2001; Welt et al. 2003.

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