Production of a mouse monoclonal IgM antibody that targets the carbohydrate Thomsen-nouveau cancer antigen resulting in in vivo and in vitro tumor killing

Abstract

The construction of a tumor-associated carbohydrate antigen-zwitterionic polysaccharide conjugate, Thomsen-nouveau-polysaccharide A1 (Tn-PS A1, where Tn = D-GalpNAc), has led to the development of a carbohydrate binding monoclonal antibody named Kt-IgM-8. Kt-IgM-8 was produced via hybridoma from Tn-PS A1 hyperimmunized Jackson Laboratory C57BL/6 mice, splenocytes and the murine myeloma cell line Sp2/0Ag14 with subsequent cloning on methyl cellulose semi-solid media. This in-house generated monoclonal antibody negates binding influenced from peptides, proteins, and lipids and preferentially binds monovalent Tn antigen as noted by ELISA, FACS, and glycan array technologies. Kt-IgM-8 demonstrated in vitro and in vivo tumor killing against the Michigan Cancer Foundation breast cell line 7 (MCF-7). In vitro tumor killing was observed using an LDH assay that measured antibody-induced complement-dependent cytotoxicity and these results were validated in an in vivo passive immunotherapy approach using an MCF-7 cell line-derived xenograft model. Kt-IgM-8 is effective in killing tumor cells at 30% cytotoxicity, and furthermore, it demonstrated approximately 40% reduction in tumor growth in the MCF-7 model.

Targeting Tumor Glycans for Cancer Therapy: Successes, Limitations, and Perspectives

“…Several mAbs recognizing the Tn antigen have been generated using different immunization strategies. Some of these antibodies are CU-1 [156], Ca3638 [157], MLS128 [158], BRIC 111 [159], 5F4 [160], HB-Tn1 [161], PMH1 [162], 83D4 [163], SM3 [164], 237mAb [165], PankoMab [166], 5E5 [167], KM3413 [168], 2154F12A4 [169], GOD3-2C4 [170], Kt-IgM-8 [171], 6C5 [172], and Remab6 [173]. Although the chemical structure of the Tn determinant is known to be GalNAcα-O-Ser/Thr, its immunological definition is more complicated, and some antibodies clearly require more complex epitopes than a single Tn residue.…”

“…In addition, the mAb PODO447 reacts with an unusual terminal motif (N-acetylgalactosamine beta-1, GalNAcβ1), predominantly found on the Podocalyxin (Podxl) protein core [176], a glycomotif rarely found in normal eukaryotic cells. Although some proteins are able to recognize a single Tn determinant, such as mAb Kt-IgM-8 [171] and plant lectins (VVLB4 and Salvia sclarea) [163,177], other anti-Tn monoclonal antibodies require at least two consecutive Tn residues for binding, such as MLS128 [178], 83D4 [163], KM3413 [168], and Remab6 [173]. This fact may be related to a higher specificity in cancer cell recognition.…”

“…In vivo experiments showing a reduction in tumor growth mediated by anti-Tn antibodies can be explained by different mechanisms: (i) antibody-dependent cellular cytotoxicity (ADCC) [20,166,168,170,182,183]; (ii) antibody-induced complement-dependent cytotoxicity (CDC) [171]; (iii) inhibition of cancer cell adhesion to lymphatic endothelium [169]; and (iv) direct blocking of receptor signaling, such as epidermal growth factor receptor and insulin-like growth factor I receptor [184]. These different mechanisms may be strongly dependent not only of the antibody class but also of the fine specificity of each antibody.…”

Chemical synthesis and immunological evaluation of entirely carbohydrate conjugate Globo H-PS A1

“…The anti-GD2 Ab, unituxin, was granted FDA approval in 2015 for the treatment of neuroblastomas. 21,22 There are several anti-TACA mAbs in preclinical/clinical trial for example mAbs to GD3, 23,24 GM2, 25 fucosyl-GM1, 26 Lewis Y, 27,28 Tn, 29,30 STn 31,32 and TF. 33 The TACAs are viable targets for immunotherapy, however, developing a qualied therapeutic anti-TACA mAb for specic types of cancer can be extremely challenging.…”

“…1a) in which all conjugates were able to induce specic adaptive immunity (target ID), production of high affinity IgG antibodies (target specicity) and tumor cell lysis (function). 29 Globo H (1 and 2) is a TACA overexpressed on a variety of tumor cells but scarcely found on healthy tissue. 52 The Globo H-PS A1 (3) construct is a continuing effort in our research group aimed at generating TACA-specic adaptive immunity and bypassing immunological challenges with protein carriers.…”