Challenges in Antibody Development against Tn and Sialyl-Tn Antigens

Abstract

The carbohydrate antigens Tn and sialyl-Tn (STn) are expressed in most carcinomas and usually absent in healthy tissues. These antigens have been correlated with cancer progression and poor prognosis, and associated with immunosuppressive microenvironment. Presently they are used in clinical trials as therapeutic vaccination, but with limited success due to their low immunogenicity. Alternatively, anti-Tn and/or STn antibodies may be used to harness the immune system against tumor cells. Whilst the development of antibodies against these antigens had a boost two decades ago for diagnostic use, so far no such antibody entered into clinical trials. Possible limitations are the low specificity and efficiency of existing antibodies and that novel antibodies are still necessary. The vast array of methodologies available today will allow rapid antibody development and novel formats. Following the advent of hybridoma technology, the immortalization of human B cells became a methodology to obtain human monoclonal antibodies with better specificity. Advances in molecular biology including phage display technology for high throughput screening, transgenic mice and more recently molecularly engineered antibodies enhanced the field of antibody production. The development of novel antibodies against Tn and STn taking advantage of innovative technologies and engineering techniques may result in innovative therapeutic antibodies for cancer treatment.

Keywords: Sialyl Tn antigen; Tn antigen; antibody production; immune response; therapeutic antibodies.

Figure 1

Pathways of Thomsen-Friedenreich antigens biosynthesis. First, GalNAc is transferred to a serine or threonine residue in a polypeptide by peptidyl-N-acetylgalactosaminyltransferases (GalNAc-T). GalNAcα1-Ser/Thr, i.e., the Tn antigen, is then converted by T-synthase to Galβ1-3GalNAcα1-Ser/Thr, i.e., the T antigen or core-1 structure. The Tn antigen can be also sialylated by ST6GalNAc-I forming Neu5Acα2-6GalNAcα1-Ser/Thr, i.e., the sialyl-Tn. GalNAcα1-Ser/Thr can also be converted to core-3 structures by the β3GnT-6. T antigen is converted to core-2 structures by C2GnT-1, -2, and -3 or can be sialylated by ST3Gal-I, originating the sialyl-T antigen. Moreover, sialyl-T antigen can be sialylated by ST6GalNAc-I originating disialyl-T antigen.

Figure 2

Simplified depiction of tumor antigen recognition and the induction of humoral immune responses. Upper panel—Glycan antigens can be directly recognized by B cells and the cross-linking of the B cell receptors lead to IgM secretion through T cell-independent B cell activation. Lower panel—Tumor antigens can be endocytosed by antigen presenting cells, such as dendritic cells, or B cells and then presented to helper T cells (Th) cells thus leading to T cell activation and subsequent T cell-dependent B cell activation. Adapted from [46].

Figure 3

Schematic representation of an immunoglobulin G (IgG) mAb structure. The IgG molecule is composed by constant (C) and variable (V) domains for each light (L) or heavy (H) chain. The heavy chain comprises three constant domains (C_H) and one variable (V_H), and the light chain contains one constant (C_L) and one variable (V_L) domain. This molecule can be divided into F_c and F_ab regions in which the latter includes the variable regions, called complementarity-determining regions (CDRs).

Figure 4

Main functions of therapeutic mAb. mAb can induce antibody dependent cell-mediated cytotoxicity (ADCC) by inducing the release of cytotoxic granules in effector cells (e.g., NK cells). It can also induce complement dependent cytotoxicity (CDC), which starts with the binding of C1q to the antibody triggering the complement cascade. mAb can induce apoptosis as well by activating caspases. In addition, mAb can block receptor/ligand interactions preventing signaling cascade activation, as well as specifically deliver drugs into tumor cells.

Figure 5

Schematic representation of the hybridoma technology. This technology involves the initial step of mice immunization with desired antigen, followed by the recovery of B cells from mice spleen and consequent fusion with myeloma cells. After that, hybridoma cells are selected in HAT medium and clones are screened according to the reactivity of their supernatants against the antigen. Finally, the selected clones are further cloned and expanded in order to obtain monoclonal antibodies with the desired specificity.

Figure 6

Schematic representation of the phage display technology. This technique includes three main steps that begin with antibody phage library construction and antibody fragments’ display onto the phage surface, followed by several rounds of selection against the target antigen (termed panning cycle) and lastly, clone isolation and subsequent screening for fragments with desired specificity.