Glycoprotein CA19.9-specific monoclonal antibodies recognize sialic acid–independent glycotope

Data of printed glycan array

Printed glycan array (PGA)^6,7 consisted of ~400 synthetic glycans, mostly fragments of glycoprotein and glycolipid carbohydrate chains. The array included the main fragment of the glycan of CA19.9 antigen, SiaLe^A, in the form of tetrasaccharide Neu5Acα2–3Galβ1–3(Fucα1–4) GlcNAc and pentasaccharide Neu5Acα2–3Galβ1–3(Fucα1–4) GlcNAcβ1–3Galβ. Natural glycoprotein CA19.9 (isolation and characterization are presented in Supplementary Data) was not printed because the difference (uncontrollable) in degree of immobilization could distort real ratio in affinities of low molecular weight (MW) glycans versus high MW glycoprotein. Concentration of the Abs was preselected to observe maximum signal close to possible upper relative fluorescence unit (RFU) value (~70,000). Data are shown in Figures 1 and 2. Notably, both mAbs do not bind SiaLe^A glycans. mAb 58/1 binds desialylated form, Le^A, whereas mAb 58/2 does not. mAb 58/2 was found to have more restricted epitope specificity. It binds only two related glycans (both are normal components of breast milk), whereas 58/1 is capable of binding to five glycans with rather different structure. However, all glycans contain the same common motif—Galβ1–3GlcNAcβ (Le^C), marked in bold in the figures. Notably, neither Le^C disaccharide as is, without substituents, nor most common naturally occurring Le^C-terminated tetrasaccharide Galβ1–3GlcNAcβ1–3Galβ1–3Glc (LNT) are positive in the PGA. Taking into account all the mentioned features, one can reconstruct glycotope for the Abs like “Galβ1–3GlcNAcβ obligatory containing an additional Ab-contacting fragment,” apparently hydrophobic, like benzyl group, or methyl group of fucose or N-acetylglucosamine residues. One ligand, Galβ1–3GlcNAcβ1–3Galβ1–4Glc, drops out of this regularity, so another “reconstruction” of real epitope could be done, namely, “Galβ1–3GlcNAcβ with distorted/constrained conformation due to a bulky substituent neighboring to Gal residue.” Although both reconstructions are well compatible to each other.

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Figure 1.

Top rank glycan ligands for (a) mAb 58/1, conc. 6 µg/mL and (b) mAb 58/2, conc. 4 µg/mL in PGA (full data of the PGA assay are presented in S6).

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Figure 2.

Direct binding of mAbs to 19-9 glycoprotein: 58/1 (black square), 58/2 (red circle).

Data of binding to PAA-conjugated glycans (enzyme-linked immunosorbent assay)

To make sure that all the observed mAbs–glycan interactions were specific, additional experiments on concentration dependence were done. Since PGA analysis is not convenient for the study of concentration dependence, we further confirmed PGA data with regular enzyme-linked immunosorbent assay (ELISA), where coating antigens were soluble polyacrylamide (PAA) conjugates of the glycans.^8,9 We selected glycans demonstrated affinity in PGA, as well as related molecules, for ELISA experiments. One of them, Galβ1–3GlcNAcβ1–3GalNAcα (Le^C-Tn), absent in PGA, was synthesized anticipating its unusual conformational properties comparing to disaccharide Le^C, and its possible tackle to cancer-associated glycans. Glycoprotein CA19.9 was also included in the list of antigens for ELISA. Data are shown in Figure 2. The results of direct binding tests for the total set of PAA are presented as histograms in Figure 3. For convenience of data comparison, absorbance values characterizing binding of mAbs with PAA glycoconjugates are given only for one of the series of consecutive dilutions of Abs used in the experiment: 1:1.6 × 10⁴ for 58/1 mAbs and 8 × 10³ for 58/2 mAbs.

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Figure 3.

Direct binding of mAbs to PAA glycoconjugates: (a) 58/1 mAbs (150 ng/mL), no binding to Galα1–3GlcNAcβ-PAA, β Fuc isomer of Le^A-PAA, 3’SiaLe^C (b) 58/2 mAbs (156 ng/mL), no binding to Galα1–3GlcNAcβ-PAA, Le^C3′αLN-PAA, Le^C-PAA, SiaLe^C-PAA, 3′SiaLe^C-PAA, 6′-O-SuLe^C-PAA, (Galβ)₂-3,4GlcNAc-PAA, β Fuc isomer of Le^A-PAA.

As in PGA assay, Ab 58/2 was found to be more narrowed, with top rank antigen found to be natural glycoprotein CA19.9, which demonstrated concentration dependence. The dependence curve for mAb 58/1 was more close to classical shape possessing well-expressed plateau. As in PGA, no significant binding was observed with SiaLe^A tetrasaccharide. Generally, the data of ELISA confirmed the results of PGA, although the coincidence was not complete. The most pronounced seems to be rather good binding of mAb 58/1 (but not of mAb 58/2) to disaccharide Le^C devoid of any additional substituents. We did not include both ligands that demonstrated the highest affinity toward 58/2, that is, nonasaccharide MF(1–3)iLNO and heptasaccharide MFLNH III, and therefore, Le^C3′LN (low binder in PGA) and Le^C-Tn (absent in PGA) apparently exhibited maximum reactivity.

Inhibitory ELISA

To confirm specificity of monoclonal binding, especially to unusual Bn-substituted glycan, we performed ELISA in an inhibitory version, using CA19.9 as coating antigen. The obtained data (Figure 4) are in accordance with direct binding, particularly high binder for mAbs 58/1 antigen 6BnLe^C appears to be a good inhibitor; similarly high binder for mAbs 58/2 antigen Le^C-Tn appears to be a good inhibitor in inhibitory ELISA. The same range of 50% inhibition concentration for 6BnLe^C and Le^Cβ3′LN (mAbs 58/1) also argues in favor of specific binding rather than just hydrophobic interaction of benzyl-substituted molecule. At the same time, rather high concentration of 50% inhibition for the top rank antigens says that true cognate antigens for both Abs are more complicated/extended than all the probes used by us in our assays.

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Figure 4.

Inhibition of mAbs (1:10⁴) binding to 19-9 by free (unconjugated) glycans: 58/1 mAbs and 6BnLe^C (red circle), Le^Cβ3’LN (green triangle); 58/2 mAbs and Le^C-Tn (black square). No inhibition in case 58/1mAbs and Le^C-Tn (3.5 mM); 58/2 mAbs and 6BnLe^C (2.1 mM).

Histochemistry

Immunohistochemical staining (Supplementary Material 8) of breast cancer tissue was performed with 58/1 Abs for five patients. The reactivity pattern was not tumor-specific; staining was observed in both normal and malignant cells; the only difference was found to be constant apical membranous expression in normal duct epithelial cells. The intensity of the staining in all cases was moderate, with no significant difference from case to case. The key difference between 58/1 and natural Abs against Le^C is reactivity of 58/1 with normal breast ducts’ epithelium (moderate intensity, membranous cytoplasmic, with accentuation of apical membrane) and smooth myocytes of breast areola.

Reagents

Horseradish peroxidase (HRPO) labeled anti-Igs were obtained from Southern Biotechnology Associates Inc. (USA). Biotin-labeled goat-anti-mouse IgG + M were obtained from Thermo Fisher (USA), and Alexa555-labeled streptavidin were obtained from Life Science (USA). Tween 20, phosphate-buffered saline (PBS; tablets), and bovine serum albumin (BSA) were obtained from Sigma (USA). All other chemicals were analytical grade from Fluka (Switzerland) or Merсk (Germany). MaxiSorp 96-well microtiter immunoplates were obtained from Nunc (Germany). PAA-conjugated glycans and their spacered forms were obtained from Lectinity (Russia). Microchips were produced by Semiotik LLC (Russia).

Study of mAbs’ specificity by ELISA

Study of mAbs’ specificity by microchip

Microarrays on glass microscope slides containing 380 glycans (50 mM solutions, at eight replicates each) were produced by Semiotik LLC (Moscow, Russia); 6 µg/mL solution of 58/1 and 4 µg/mL solution of 58/2 Abs in PBS-1% containing 1% BSA were applied to array (pretreated with PBS-0.1% for 15 min) and incubated in a humidified chamber for 1.5 h at 37°C. Then the microarrays were washed with PBS-0.1% and incubated with the 20 µg/mL solution of biotin-labeled secondary goat-anti-mouse IgG + M in PBS-0.1% under the same conditions. After washing with PBS-0.001%, the 1 µg/mL solution of Alexa555-labeled streptavidin in PBS-0.1% was added and arrays were incubated in a humidified chamber for 45 min at 23°C. After washing with PBS-0.05% and deionized water, the arrays were scanned on a InnoScan AL 1100 scanner (Innopsys, France) using the excitation wavelength of 543 nm. The obtained data were processed using ScanArray Express 4.0 software and the fixed 80-µm-diameter circle method as well as Microsoft Excel software. Data are reported as median RFUs of replicates. Median deviation was measured as interquartile range. A signal the fluorescence intensity of which exceeded the background (i.e. the signal from the surface which is not containing a ligand) value by a factor of 5 was considered as significant.