Characterization of a variety of neutralizing anti-heparin-binding epidermal growth factor-like growth factor monoclonal antibodies by different immunization methods

Figures & data

Table 1. Summary of anti-HB-EGF mAb generation by mouse strain and immunogen

Immunization	Total	HB-EGF binder	Neutralizing mAb
KLH/BALB/c^a	17,952	1,018 (5.7%)	19 (0.11%)
KLH/sHB-EGF/BALB/c^b	2,640	207 (7.8%)	24 (0.91%)
KLH/cell/BALB/c^c	5,280	589 (11.2%)	33 (0.63%)
KLH/C57BL^d	1,670	30 (1.8%)	1 (0.06%)
KLH/C3H^e	2,640	210 (8.0%)	2 (0.08%)
KLH/CD1^f	5,720	754 (13.2%)	41 (0.72%)
BSA/CD1^g	4,576	529 (11.6%)	90 (1.97%)
Total	40,478	3,337 (8.2%)	210 (0.52%)

The number of hybridoma clones of mAb for each immunization is shown. The production rates are noted in parentheses. ^a-cBALB/c immunization with ^aKLH-conjugated sHB-EGF; ^bthe KLH-conjugate and a final boost of sHB-EGF; ^cco-immunization of the KLH-conjugate and proHB-EGF-expressing cells; ^dC57BL immunization with the KLH-conjugate; ^eC3H immunization with the KLH-conjugate; ^f,gCD1 immunization with ^fthe KLH-conjugate; ^gBSA-conjugated sHB-EGF.

Figure 1. Neutralizing activity of anti-HB-EGF mAbs against sHB-EGF functions. (A) Neutralizing activity of anti-HB-EGF mAbs against sHB-EGF-induced EGFR phosphorylation in SK-OV-3 cells. The plots represent the IC₅₀ values of 146 mAbs used in the epitope binning study (Fig. 3). The horizontal line indicates the median IC₅₀ value. (B) Neutralizing activity of anti-HB-EGF mAbs against sHB-EGF-induced colony formation in RMG-I cells. We used the 89 mAbs with IC₅₀ values of less than 5 × 10⁻⁹ M in the EGFR phosphorylation assay, and this plot represents the IC₅₀ values of the 83 mAbs with IC₅₀ values of less than 1 × 10⁻⁷ M. The horizontal line indicates the median IC₅₀ value. (C) Correlation of neutralizing activities of anti-HB-EGF mAbs against sHB-EGF-induced EGFR phosphorylation and colony formation. Each dot represents the IC₅₀ values of 83 mAbs with an IC₅₀ value of less than 1 × 10⁻⁷ M in the colony formation assay (B). The data were analyzed by the Spearman’s test (two-tailed). The Spearman correlation was r = 0.6730 (p < 0.0001).

Figure 2. Binding activities of neutralizing anti-HB-EGF mAbs to HB-EGF. Binding activity of anti-HB-EGF mAbs to (A) human sHB-EGF, (B) mouse sHB-EGF and (C) rat sHB-EGF. The binding activities were determined by ELISA. The plots represent EC₅₀ values of 146 mAbs used in the epitope binning study (Fig. 3). The horizontal line indicates the median EC₅₀ value. (D) Binding activity of anti-HB-EGF mAbs to proHB-EGF was measured by flow cytometry. The plots represent median fluorescence intensity (MFI) ratio of the 146 mAbs used in the epitope binning study (Fig. 3). The MFI ratio of anti-HB-EGF mAb to a control sample was used as the measure of binding activity to proHB-EGF. The horizontal line indicates the median MFI ratio.

Figure 3. Epitope binning study of anti-HB-EGF mAbs. The competitive binding of all mAb pairs to sHB-EGF was measured in the FMAT assay. Binding of biotinylated anti-HB-EGF mAb to sHB-EGF was detected in the presence of a label-free anti-HB-EGF mAb. Horizontal and vertical axes indicate unlabeled and biotinylated mAbs, respectively. Data were analyzed by Ward’s method with Euclidean distance. Dendrogram on the left axis represents the similarity of each mAb in the competitive binding pattern to sHB-EGF. The color scale from 0 to 100 shows the competitive binding of the 2 mAbs.

Figure 4. Difference of epitope bin population by mouse strain and immunogen type. Epitope bin populations were analyzed based on the immunization method used to generate the mAbs. ^a-cBALB/c immunization with ^aKLH-conjugated sHB-EGF; ^bKLH-conjugate and a final boost of sHB-EGF; ^cco-immunization of the KLH-conjugate and proHB-EGF-expressing cells; ^d,eCD1 immunization with ^dKLH-conjugate; ^eBSA-conjugated sHB-EGF.