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Intracellular tumor-associated antigens are targeted by antibodies known as T-cell receptor mimic antibodies (TCRm-Abs), which recognize T-cell epitopes with better stabilities and higher affinities than T-cell receptors. However, TCRm-Abs have been proven difficult to produce using conventional techniques. Here, we developed TCRm-Abs that recognize the survivin-2B-derived nonamer peptide, AYACNTSTL (SV2B 80-88 ), presented on HLA-A*24 (SV2B 80-88 /HLA-A*24) from immunized mice by using a fluorescence-activated cell sorting-based antigen-specific plasma cells isolation method combined with a high-throughput single-cell-based immunoglobulin-gene-cloning technology. This approach yielded a remarkable efficiency in generating candidate antibody clones that recognize SV2B 80-88 /HLA-A*24. The screening of the antibody clones for their affinity and ability to bind key amino-acid residues within the target peptide revealed that one clone, #21-3, specifically recognized SV2B 80-88 /HLA-A*24 on T2 cells. The specificity of #21-3 was further established through survivin-2B-positive tumor cell lines that exogenously or endogenously express HLA-A*24. A bispecific T-cell engager comprised of #21-3 and anti-CD3 showed specific cytotoxicity towards cells bearing SV2B 80-88 /HLA-A*24 by recruiting and activating T-cells in vitro. The efficient development of TCRm-Ab overcomes the limitations that hamper antibody-based immunotherapeutic approaches and enables the targeting of intracellular tumor-associated antigens.

Threonine phosphorylation accounts for 10% of all phosphorylation sites compared with 0.05% for tyrosine and 90% for serine. Although monoclonal antibody generation for phospho-serine and -tyrosine proteins is progressing, there has been limited success regarding the production of monoclonal antibodies against phospho-threonine proteins. We developed a novel strategy for generating phosphorylation site-specific monoclonal antibodies by cloning immunoglobulin genes from single plasma cells that were fixed, intracellularly stained with fluorescently labeled peptides and sorted without causing RNA degradation. Our high-throughput fluorescence activated cell sorting-based strategy, which targets abundant intracellular immunoglobulin as a tag for fluorescently labeled antigens, greatly increases the sensitivity and specificity of antigen-specific plasma cell isolation, enabling the high-efficiency production of monoclonal antibodies with desired antigen specificity. This approach yielded yet-undescribed guinea pig monoclonal antibodies against threonine 18-phosphorylated p53 and threonine 68-phosphorylated CHK2 with high affinity and specificity. Our method has the potential to allow the generation of monoclonal antibodies against a variety of phosphorylated proteins.