Monoclonal antibodies toward different Tn-amino acid backbones display distinct recognition patterns on human cancer cells. Implications for effective immuno-targeting of cancer

Abstract

The Tn antigen (GalNAcα-O-Ser/Thr) is a well-established tumor-associated marker which represents a good target for the design of anti-tumor vaccines. Several studies have established that the binding of some anti-Tn antibodies could be affected by the density of Tn determinant or/and by the amino acid residues neighboring O-glycosylation sites. In the present study, using synthetic Tn-based vaccines, we have generated a panel of anti-Tn monoclonal antibodies. Analysis of their binding to various synthetic glycopeptides, modifying the amino acid carrier of the GalNAc(*) (Ser* vs Thr*), showed subtle differences in their fine specificities. We found that the recognition of these glycopeptides by some of these MAbs was strongly affected by the Tn backbone, such as a S*S*S* specific MAb (15G9) which failed to recognize a S*T*T* or a T*T*T* structure. Different binding patterns of these antibodies were also observed in FACS and Western blot analysis using three human cancer cell lines (MCF-7, LS174T and Jurkat). Importantly, an immunohistochemical analysis of human tumors (72 breast cancer and 44 colon cancer) showed the existence of different recognition profiles among the five antibodies evaluated, demonstrating that the aglyconic part of the Tn structure (Ser vs Thr) plays a key role in the anti-Tn specificity for breast and colon cancer detection. This new structural feature of the Tn antigen could be of important clinical value, notably due to the increasing interest of this antigen in anticancer vaccine design as well as for the development of anti-Tn antibodies for in vivo diagnostic and therapeutic strategies.

Fig. 1

Analysis of MAb specificities by surface plasmon resonance measurements. Binding analysis of different anti-Tn MAbs: a 83D4 (25 nM), b 2A10 (100 nM), c 6E11 (100 nM), d 9A7 (100 nM), and e 15G9 (12 nM) was performed on biosensor surfaces with immobilized biotinylated glycopeptides. These glycopeptides display different tri-Tn clusters (S*T*T*, T*T*T*, or S*S*S*) linked to an irrelevant poly-glycine peptide different from the immunogen PV peptide (see “Methods”). The biotinylated unglycosylated peptide (STT) was used as the negative control. RU resonance units

Fig. 2

Anti-Tn MAbs differentially recognize native forms of Tn on tumor cell lines. Anti-Tn MAbs were tested for the binding to Tn-expressing Jurkat, MCF-7, and LS74-T cells, by flow cytometry, using PE conjugated anti-mouse IgG antibodies, except for 83D4 MAbs for which anti-mouse IgM was used. Filled gray histograms correspond to the secondary reagent alone and black lines to indicated MAb

Fig. 3

Immunohistochemical evaluation of anti-Tn MAbs in breast tumors. a Anti-Tn MAbs 83D4, 2A10, 6E11, 9A7, and 15G9 were probed against a primary breast tumor (magnification ×40). The immunohistochemical staining (score 0–300) was assessed as the product of the intensity (0, 1, 2, or 3) and the percentage of positive cells. Staining intensity and immunohistochemical score assigned to each sample were 83D4 (3/300), 2A10 (2/160), 6E11 (1/30), 9A7 (1/70), and 15G9 (0/0). b Representative picture of positive (6E11) or negative (9A7) staining of a primary breast tumor. Magnification ×100. Staining intensity and immunohistochemical scores were 6E11 (3/300) and 9A7 (0/0)

Fig. 3

Immunohistochemical evaluation of anti-Tn MAbs in breast tumors. a Anti-Tn MAbs 83D4, 2A10, 6E11, 9A7, and 15G9 were probed against a primary breast tumor (magnification ×40). The immunohistochemical staining (score 0–300) was assessed as the product of the intensity (0, 1, 2, or 3) and the percentage of positive cells. Staining intensity and immunohistochemical score assigned to each sample were 83D4 (3/300), 2A10 (2/160), 6E11 (1/30), 9A7 (1/70), and 15G9 (0/0). b Representative picture of positive (6E11) or negative (9A7) staining of a primary breast tumor. Magnification ×100. Staining intensity and immunohistochemical scores were 6E11 (3/300) and 9A7 (0/0)

Fig. 4

Immunohistochemical evaluation of anti-Tn MAbs in colon tumors. Anti-Tn MAbs 83D4, 2A10, 6E11, 9A7, and 15G9 were probed against a primary colon tumor or normal tissue. Magnification ×400. Staining intensity and immunohistochemical score assigned to each sample were 83D4 (3/294), 2A10 (2/196), 6E11 (2/190), 9A7 (1/95), and 15G9 (1/90)

Fig. 5

Human tissue microarray analysis of Tn antigen expression in breast tumors. A representative case stained by MAbs 83D4, 2A10, 6E11, and 9A7 and negative for MAb 15G9. Magnification ×40. Staining intensity and immunohistochemical score assigned to each sample were 83D4 (3/300), 2A10 (2/180), 6E11 (2/200), 9A7 (1/40), and 15G9 (0/0)

Fig. 6

Human tissue microarray analysis of Tn antigen expression in colon tumors. A representative case showing staining both in cancer cell and in the secretory material (MAb 83D4), staining only in cancer cells (MAbs 2A10, 6E11, and 9A7), and absence of staining (MAb 15G9). Magnification ×100. Staining intensity and immunohistochemical score assigned to each sample were 83D4 (3/300), 2A10 (2/120), 6E11 (2/140), 9A7 (1/30), and 15G9 (0/0)