A combination of in vitro techniques for efficient discovery of functional monoclonal antibodies against human CXC chemokine receptor-2 (CXCR2)

Figures & data

Figure 1. Strategy for monoclonal antibody development by naïve antibody phage display library panning and B cell hybridoma technique.

Figure 2. Identification of IL-8 binding sites on CXCR2. (A) Binding of IL-8 to array of > 3,600 different peptide constructs. The peptide arrays were challenged with biotinylated IL-8 and detected with streptavidin-HRP. (B) Cartoon of CXCR2. Seven transmembrane domains (cylinders) are connected by 3 intra cellular loops (not labeled) and 3 extracellular loops (ECL1, ECL2 and ECL3) with 2 disulfide bonds (solid lines), between ECL3 and N-terminus, and between ECL1 and ECL2, respectively. Black dots represent the inferred binding positions of IL-8. (C) Peptide reconstruction of the extracellular regions of hCXCR2. N-terminus (DSFEDFWKGEDLSNYSYSSTLPPFLLDAAPCEPESLEINK) (black structure) and ECL3 (DTLMRTQVIQETCERRNHIDR) (dark gray arch) are connected by a disulfide bond (antigen A) or a CLIPS™ moiety (antigen B). The biotin moiety (gray square), for antibody phage display library panning, is located at the C-terminal side of the N-terminus.

Table 1A. scFvs isolated from panning naïve antibody phage display libraries

	2 panning rounds (peptide/peptide)		3 panning rounds (peptide/peptide/cell)
	no. of unique^1 scFv's binding to		no. of unique^1 scFv's binding to
antigen	antigen	hCXCR2^2	antigen	hCXCR2^2
A	32	9 (28%)^3	31	17 (55%)
B	41	3 ( 7%)	28	23 (82%)

¹Uniqueness initially assessed by fingerprinting, subsequently confirmed by sequencing. ²Binding to HEK293 cells stably expressing huCXCR2, but not to the parental cell line.³Percentage subpopulation antigen binding clones cross-reacting with CXCR2 overexpressing cells, but not the parental cells.

Table 1B. Antibodies from mouse hybridomas, elicited with antigen A

no. of clones binding to antigen	hCXCR2^1
15	7

¹Binding to HEK293 cells stably expressing huCXCR2, but not to the parental cell line.

Figure 3. Top Panel: Inhibition of IL-8 (A and C) and Gro-α (B and D) induced ß-arrestin recruitment in hCXCR2-HEK cells (Tango™ GPCR Assay System, Invitrogen) by IgGs converted from scFvs (A and B): 4138.51 (open square), 4081.73 (solid circle), 4138.73 (filled diamond), 4138.06 (asterisk), 4138.27 (open triangle), 4138.39 (open circle), 4138.14 (solid triangle) 4138.54 (solid star), and 4138.40 (open diamond). Murine IgGs (C and D): mAb27 (solid diamond), mAb5 (solid circle), mAb9 (solid square), mAb17 (open square), mAb7 (open circle), mAb21 (open diamond) and mAb19 (solid triangle). Each curve is based on 2 independent measurements except for panel A which is based on 4 independent measurements. The error bars represent the standard deviations. Bottom panel: Using GraphPad Prism software (Software MacKiev) the top values of each dataset were constrained at 100% and the bottom values of each dataset were shared. In all panels the Hill slopes were constrained at -1.000. IgGs converted from scFvs (A and B): 4138.51 (a), 4081.73 (b), 4138.73 (c), 4138.06 (d), 4138.27 (e), 4138.39 (f), 4138.14 (g) 4138.54 (h), and 4138.40 (i). Murine IgGs (C and D): mAb27, mAb5, mAb9, mAb17, mAb7, mAb21 and mAb19. Each curve is based on 2 independent measurements except for panel A which is based on 4 independent measurements.

Table 2. Binding to native CXCR2 and functional testing of recombinant IgGs (converted from scFvs) and murine monoclonal antibodies

		MFI ratio CXCR2/parental binding
		[mAb] (μg/ml)			IC50 ligand inhibition (nM)
panning sequence	IgG^1	0.01	0.1	1	IL8	Gro-a
A/A/cells	4138.51	6	45	161	0.3	0.2
A/A	4081.73	4	13	9	0.4	0.3
A/A/cells	4138.73	2	8	18	3	1.2
B/B/cells	4138.06	2	11	44	5	10
A/A/cells	4138.27	1	4	4	9	8
A/A/cells	4138.39	1	4	14	10	4
A/A/cells	4138.14	n.d.	1	2	67	29
B/B/cells	4138.54	2	6	31	84	69
B/B/cells	4138.40	2	6	20	91	20
A/A/cells	4138.38	n.d.	17	37	> 1000	> 1000
A/A/cells	4139.86	n.d.	1	4	> 1000	> 1000
B/B/cells	4138.18	n.d.	1	4	> 1000	> 1000
B/B/cells	4138.89	n.d.	1	2	> 1000	> 1000
antigen	murine mAb
A	mAb 27	2	8	80	18	19
A	mAb 5	1	6	72	84	72
A	mAb 9	1	7	47	152	134
A	mAb 7	1	3	30	> 1000	> 500
A	mAb 17	5	31	237	> 1000	> 500
A	mAb 21	1	3	23	> 1000	> 1000
A	mAb 19	1	1	3	> 1000	> 1000

n.d. = not done.¹All IgGs (converted from scFvs) originate from peptide/peptide/cells panning, except IgG 4081.73 which originates from peptide/peptide panning.

Table 3. Epitope mapping of recombinant IgGs (converted from scFv) and murine monoclonal antibodies

	ELISA^1		epitope mapping CXCR2 N-terminus^5
	[mAb](ng/ml)^2
IgG	Nterm-N^3	Nterm-C^4	MEDFNMESDSFEDFWKGEDLSNYSYSSTLPPFLLDAAPCEPESLEINK
4138.51	12	> 1000	ESDSFEDFWK
4081.73	6	> 1000	FEDFWK     SSTLPPFLLDA
4138.73	4	> 1000	DSFEDFWK
4138.06	8	> 1000	FNMESDSFEDFWK    YSSTLPPFLLD
4138.39	9	> 1000	FEDFWK
4139.86	37	> 1000	MESDSFEDFWKGEDLSN
4138.38	8	> 1000	FEDFWKGEDLSNYSYSSTLPPFLLDA
4138.18	22	> 1000	ESDSFEDFWKGE LSNYSYSSTLPPFLLD
murine mAb
mAb 27	> 1000	4	FEDFWKGEDLSN     LPPFLLDAAP
mAb 5	> 1000	3	FEDFWKGEDL       LPPFLLDAA
mAb 9	> 1000	3	FEDFWKGEDL       PPFLLDAA
mAb 7	> 1000	4	LPPFLLDAAPCE
mAb 17	7	629	DSFEDFWKGEDL       PPFLLDAAP
mAb 19	> 1000	276	PPFLLDAA
mAb 21	6	> 1000	PPFLLDAA

¹The binding concentration of all listed monoclonal antibodies was > 1000 ng/ml for linear ECL1 (biotin-Ahx-GGAASKVNGWIFGTFLAK-NH₂), cyclic ECL1 (biotin-Ahx -CASKVNGWIFGTFL C_(Acm)KC-NH2), linear ECL3 (biotin-Ahx -GGDTLMRTQVIQETAERRNHIDR-NH₂) and cyclic ECL3 (biotin-Ahx -GGhCDTLMRTQVIQETAERRNHIDRGhC-NH₂) representing peptides. The cyclic peptides were cyclized by CLIPS™ moieties.

²[mAb] (ng/ml) at 4-fold background OD.

³Nterm-N = linear N-terminal part of the CXCR2 N-terminus: biotine-Ahx-GGMEDFNMESDSFEDFWKGEDLSNYSYSS-NH2 (Ahx = alpha hexanoic acid).

⁴Nterm-C = linear C-terminal part of the CXCR2 N-terminus: biotine-Ahx-GGSYSSTLPPFLLDAAPAEPESLEINK-NH2.

⁵Epitope mapping included 51 overlapping 16-mers peptide array comprising the N-terminus, ECL1 (data not shown), ECL2 (data not shown), and ECL3 (data not shown) domains of hCXCR2. Dominant epitopes are indicated in bold, weak epitopes in regular.

Pierce KL, Premont RT, Lefkowitz RJ. Seven-transmembrane receptors. Nat Rev Mol Cell Biol 2002; 3:639-50; PMID:12209124; http://dx.doi.org/10.1038/nrm908

 PubMed Web of Science ®Google Scholar