Purified human anti-Tn and anti-T antibodies specifically recognize carcinoma tissues

Described in several epithelial cancer cells, Tn- (GalNAcα1-O-Ser/Thr) and T- (Galβ3GalNAcα1-O-Ser/Thr) antigens are examples of tumor-associated antigens. Increased expression of Tn- and T-antigens is associated with tumor invasion and metastasis, and patients with high concentration of anti-Tn and anti-T antibodies have a more benign evolution of pathology. Asialofetuin (ASF) and ovine submaxillary mucin (OSM) are two glycoproteins that expose T- and Tn-antigen, respectively. In this work, using ASF or OSM we affinity-purified anti-T and anti-Tn antibodies from normal human plasma and tested their ability to specifically recognize tumor human tissues. Whereas purified anti-T antibodies (purity degree increase of 127-fold, and 22% recovery) were mainly IgG, for purified anti-Tn antibodies (purity degree enhancement of 125-fold, and 26% yield) the IgM fraction was predominant over the IgG one. IgG2 subclass was significantly enriched in both purified antibody samples. Purified antibodies did not bind normal human tissue (0/42), although recognized malignant tissues from different origin such as colon carcinoma (11/77 by anti-Tn; 7/79 by anti-T), breast carcinoma (10/23 by anti-Tn; 7/23 by anti-T), and kidney carcinoma (45/51 by anti-Tn; 42/51 by anti-T). Our results suggest that purified human anti-Tn and anti-T antibodies have a potential as anti-tumor therapeutic agents; restoring their levels in human sera could positively affect the evolution of patients with epithelial tumor pathologies.

Normal human sera contain multiple antibodies recognizing specific glycan residues 7 , and different hypothesis attempt to explain the origin of natural anti-glycan antibodies 8 . Natural anti-Tn and anti-T antibodies are present in normal human sera 9 , and studies of anti-Tn and anti-T antibodies in patients with epithelial carcinomas showed reduced levels of these anti-glycan antibodies 10 . In addition, pathology evolution of patients with high concentration of anti-Tn and anti-T antibodies is more benign 11 . These results suggest that restitution of human anti-Tn and anti-T antibodies should positively affect the evolution of patients with epithelial tumor pathologies.
Immunotherapy modulates the host's immune response to TAAs, eradicates cancer cells by reducing host tolerance to TAAs and provides protection against the disease [12][13][14] . Passive immunotherapies, like monoclonal antibodies or engineered T-cell based therapies, are targeted to tumor cells by recognizing TAAs. Several immunotherapy strategies have been tested for anti-tumor responses using monoclonal antibodies against receptor tyrosine kinases like members of the EGFR family (cetuximab, pertuzumab, and trastuzumab) 15,16 or against their ligands like VEGF (bevacizumab) 17 , involved in tumor cell proliferation or angiogenesis, respectively.
In the present study we purified two populations of antibodies (anti-Tn and anti-T) from pooled human plasma and evaluated their ability to recognize human carcinoma tissue, aiming to uncover potential applications in antineoplastic therapy.

Results
Purification of anti-glycan antibodies. Asialofetuin (ASF) and ovine submaxillary mucin (OSM) are two highly glycosylated antigens. ASF mainly exposes terminal T-antigen glycans, whereas OSM shows multiple terminal Tn-and sialyl Tn-antigens 18,19 . By immobilizing ASF and OSM in Sepharose, these terminal glycans were used as ligands for affinity chromatography purification of human antibodies. Gammaglobulin fraction from pooled human plasma was offered to immobilized antigens. After washing the columns, retained proteins were eluted and analyzed. The purity of eluted proteins was evaluated by SDS-PAGE stained with Coomassie Brilliant Blue (CBB), and immunoglobulin isotypes were identified by western blot (WB) using anti-human IgG, IgM and total immunoglobulin antibodies (Fig. 1).
SDS-PAGE stained with CBB showed highly purified proteins, with molecular weight corresponding to human immunoglobulins. WB showed that purified anti-ASF antibodies are mainly IgG with reduced proportion of IgM, whereas in the purified human anti-OSM antibodies the IgM fraction is important with respect to IgG. To know the purification degree and yield of each purified antibody, proteins were measured by bicinchoninic acid assay, and human immunoglobulins were measured by ELISA using HRP-labeled IgG anti-human total immunoglobulin antibodies (Fig. 2). Affinity chromatography for human antibodies recognizing ASF increased the purity degree 127-fold with 22% recovery (Table 1), whereas the purification of anti-OSM antibodies enhanced 125-fold the specific activity with 26% yield (Table 2). Further characterization of both purified antibodies showed a very important enrichment of IgG2 subclass (Tables 1 and 2).
Carbohydrate specificity of purified anti-glycan antibodies. The recognition of purified anti-ASF and anti-OSM antibodies to the corresponding antigen used during the affinity chromatography purification are shown in Fig. 3. Carbohydrate specificity of purified antibodies was evaluated by competitive Quadruplicates for each eluted sample were seeded in polyacrylamide gels to analyze degree of protein purity by staining with Coomassie Brilliant Blue (a), or by western blot detecting heavy chain presence of human IgG (b), human IgM (c), or human total immunoglobulins (d) by using HRP-labeled IgG anti-human IgG, or IgM or total immunoglobulins antibodies, respectively. Color reaction was developed as described in Methods.
www.nature.com/scientificreports www.nature.com/scientificreports/ ELISA using sugars related to Tn-and T-antigen. Interaction of anti-ASF antibodies was significantly inhibited with Galβ3GalNAcαBzl, whereas glucose did not induce inhibition (Table 3). Binding inhibition with Galβ3GalNAcαBzl revealed the presence of anti-T antibodies in this purified antibody sample. OSM-affinity    www.nature.com/scientificreports www.nature.com/scientificreports/ chromatography yielded anti-Tn antibodies, as showed by the important inhibition with GalNAcαBzl whereas control (glucose) did not show inhibition (Table 4).

Anti-Tn and anti-T antibodies specifically recognize human carcinoma tissues. The capacity
of human purified anti-Tn and anti-T antibodies to recognize human carcinoma tissues was analyzed by tissue micro-arrays (TMAs). Human TMAs from normal breast, kidney, colon, skin, placenta, cervix, fallopian tube, pancreas, amygdale, endometrium, small intestine, ovary, spleen, and brain tissues, as well as carcinomas from skin, colon, breast, and kidney tissues were assayed to study carcinoma recognition of purified antibodies. Representative images of human normal and malignant tissues, and human cell lines with negative and positive staining are shown in Fig. 4. Purified anti-Tn and anti-T antibodies did not recognize (0/42) normal human tissues (Fig. 5). Human melanoma tissues were not recognized (0/12) by purified anti-Tn or anti-T antibodies. Human carcinoma colon tissues were positively stained in 14% (11/77) by anti-Tn antibodies and in 8.9% (7/79) by anti-T antibodies. In addition, breast carcinoma tissue showed positive immunoreactivity in 43% (10/23) and 30% (7/23) by anti-Tn and anti-T antibodies, respectively. Table 5 shows features of human breast carcinomas. Also, human breast tumor T47D cell line showed strong positive recognition by purified antibodies (Fig. 4). Human embryonic kidney (HEK-293) cells were less immunoreactive that tumor T47D cells with purified anti-Tn and anti-T antibodies (Fig. 4). Human kidney carcinoma was the most recognized tissue by these antibodies, showing positive staining in 88% (45/51) by anti-Tn antibodies and in 82% (42/51) of carcinoma tissues by anti-T antibodies (Fig. 5). Table 6 shows characteristic of human kidney tumor tissues and the recognition of purified antibodies.

Discussion
Passive immunotherapy of patients with cancer pathologies implicate incorporation of molecules addressed against the tumor. Thus, specific molecules are used to decrease the transformation, proliferation and survival of cancer cells. Mouse monoclonal humanized antibodies (mAbs) such as obinutuzumab, rituximab and trastuzumab are examples of successful passive immunotherapies in the treatment of cancer patients 20 . However, these mAb treatments showed side effects such as ocular and neuro-ophthalmic toxicity, skin toxicities with different severities (from mild lichenoid reaction to severe toxic epidermal necrolysis), increased risk factors in development of hypogammaglobulinemia and infections (post-rituximab), and increased cumulative incidence of breast cancer brain metastases after HER2-directed therapy (trastuzumab) [21][22][23][24] . In the present work, we obtained human anti-Tn and anti-T antibodies that specifically recognize human carcinoma tissues, with potential use for restoration of anti-glycan antibody concentrations of patients presenting epithelial tumor pathologies.
Immobilized ASF and OSM allowed the purification of natural antibodies present in normal human plasma. Both purified antibodies enhanced more than 120-times the purification degree compared to plasma presence. Related to immunoglobulin isotype of purified antibodies, anti-ASF antibodies were mainly IgG whereas anti-OSM antibodies contained IgM in similar proportion to IgG. In agreement with this purification, the presence of IgM and IgG2 subclass as constitutive of natural anti-glycan antibodies has been frequently described 25,26 .  Table 3. Carbohydrate recognition of anti-T antibodies by competitive assay 1 . 1 Competitive assay using carbohydrates as inhibitors of anti-ASF antibody recognition. Different concentration of carbohydrates preincubated with anti-ASF antibodies showed the significance of sugar residue in the antigen recognition. Galβ3GalNAcαBzl, structure related to T-antigen, is important inhibitor of antibody interaction whereas glucose (Glc) has not effect (control).  www.nature.com/scientificreports www.nature.com/scientificreports/ The classical way to study glycan recognition of proteins is through competitive assays with soluble carbohydrates. This useful tool allowed us to observe that, upon purification, human natural anti-ASF antibodies recognize related Thomsen-Friedenreich structure (T antigen), and that human natural anti-OSM antibodies recognize Tn antigen. Thus, both purified antibodies showed the expected glycan recognition specificity.
The ability of these purified anti-glycan antibodies to recognize human carcinomas was studied using TMAs. Purified anti-T and anti-Tn antibodies did not interact with normal human tissues, a consistent observation since these antibodies were purified from normal individuals. This represents an advantage compared to observations from previous antibody approaches: therapeutic use for several mouse humanized monoclonal antibodies reported side effects as skin and neuro-ophthalmic toxicities, thus suggesting recognition of normal tissues 23,24 .
Purified anti-Tn and anti-T antibodies did not show capacity to recognize human melanomas, and colon carcinomas were poorly recognized. The binding ability of these antibodies improved for breast human carcinomas tissues (43% for anti-Tn antibodies and 30% for anti-T antibodies), and they were even more efficient in recognizing human kidney carcinoma tissues. Trastuzumab (anti-Her2 antibody) is present in the pharmaceutical market www.nature.com/scientificreports www.nature.com/scientificreports/ having only 3% Her2-positive immunostaining across human carcinomas, with frequencies ranging from 0.4% in hepatocellular carcinoma to 12.4% in bladder cancer 27 . The antibodies obtained herein have better recognition ability to human carcinomas than trastuzumab, thus deserving further attention.
The analysis of carcinoma Fuhrman grades related to positive recognition for the obtained antibodies showed no differences. Anti-Tn and anti-T antibodies recognized equally well carcinomas with Fuhrman degree 2 and 3 from human breast and kidney tissues. This fact shows that these antibodies could be useful for patients' treatment with early Fuhrman grades of breast and kidney carcinomas, such as degree 2.
In summary, we have outlined the production of two natural antibodies from normal human plasma. Their relevant capacity to efficiently and specifically recognize human carcinoma tissues brings hope about their potential use as antitumoral antibodies to restitute the anti-Tn and anti-T antibody concentrations in patients with carcinoma pathologies.

SDS-PAGE and western blotting. Homogeneity of purified antibodies was confirmed by SDS-PAGE and
Coomassie Brilliant Blue staining 29 . To evaluate their isotypes, purified antibodies were subjected to SDS-PAGE, and electrotransferred membranes were blocked with 1% skim milk in PBS for 60 min at room temperature (RT). HRP-labeled IgG anti-human IgG, anti-human IgM, or anti-total immunoglobulin antibodies (Sigma-Aldrich, St. Louis, USA; 1/1000 dilution in 0.05% Tween 20 in PBS) were incubated for 2 h at RT and washed with PBS. Color reaction was developed using 0.1 mg/ml 4-chloro-1-naphthol and 0.02% H 2 O 2 in methanol-TBS for 20 min, and stopped by washing with distilled water.

Measurement of total proteins and immunoglobulins.
A bicinchoninic acid assay was used to measure total proteins with BSA as the standard (Pierce, Thermo Scientific). To measure total immunoglobulin concentration, wells of microtiter plates were coated with several concentrations of purified total human immunoglobulins (Gammaglobulina-T, Laboratorio de Hemoderivados, UNC, Argentina) in 0.1 M carbonate buffer (pH 9.5) overnight at 4 °C. After blocking with PBS containing 1% BSA and 0.05% Tween 20, plates were incubated with 1:1000 HRP-conjugated IgG anti-human total immunoglobulin antibodies (Sigma-Aldrich, St. Louis, USA) in PBS-T, for 2 h at RT. Plates were washed 4x with PBS-T, and color reaction was developed using 2 mg/ml o-phenylenediamine and 0.02% H 2 O 2 in 0.1 M sodium citrate (pH 5.0), at RT for 10 min. Reaction was stopped by addition of 0.5 M sulfuric acid. Absorbance values were read at 490 nm with a microplate reader. Similarly, human IgM and IgG was measured using HRP-labeled IgG anti-human IgM and anti-human IgG antibodies, and  www.nature.com/scientificreports www.nature.com/scientificreports/ concentrations determined from standard curves of human IgG and IgM, respectively. Radial immunodiffusion for measuring human IgG1, IgG2, IgG3 and IgG4 was performed as suggested by manufacturer (The Binding Site Inc., San Diego, USA) and the concentrations were determined from standard curves.

Tissue microarrays (TMAs). All procedures, performed in accordance with Ethical Guidelines on Research
Involving Human Subjects 28 and with ethical standards as laid down in the 1964 Declaration of Helsinki and its later amendments, were approved by the Ethics Committee of CIQUIBIC-CONICET; informed consent was obtained from the patients. Paraffin-embedded tissues were selected from histological file and used in a blind-retrospective manner. Representative areas from normal tissues (n = 42), skin melanoma (n = 12) and carcinomas (colon n = 77, breast n = 23, kidney n = 51) were carefully marked on H&E-stained sections. Tissue cores of 2 mm diameter were obtained from each specimen and were precisely arrayed into a new paraffin block. 4 μm thickness sections of this new block were cut and mounted on polarized slides to future staining.