Tn-antigen (GalNAcα-Ser/Thr) is one of the most common aberrations associated with cancer progression and metastasis, and thus is an excellent target for development of cancer diagnostics and therapeutics. MLS128 monoclonal antibody (mAb), derived from a mouse immunized with human colon carcinoma cells, was reported to bind to two or three consecutive Tn-antigens (Tn2 or Tn3) with one-order higher affinity for Tn3 than for Tn2. Our recent studies demonstrated that MLS128 significantly inhibits breast and colon cancer cell growth. Molecular cloning of the variable regions of heavy (VH) and light (VL) chains revealed that the VH sequence of MLS128 shared 97% nucleotide sequence identity with the VH of 83D4 mAb, derived from breast cancer-immunized mice, which has a similar affinity for Tn2/Tn3. MLS128 single-chain antibodies (scFv) and scFv-Fc were constructed to confirm the affinity for synthetic Tn2/Tn3 peptides. Thermodynamic studies on MLS128 binding to Tn2/Tn3 revealed its unique nature of temperature-dependent binding.