A TCR mimic monoclonal antibody reactive with the “public” phospho-neoantigen pIRS2/HLA-A*02:01 complex
Tao Dao, Sung Soo Mun, Zaki Molvi, Tatyana Korontsvit, Martin G. Klatt, Abdul G. Khan, Elisabeth K. Nyakatura, Mary Ann Pohl, Thomas E. White, Paul J. Balderes, Ivo C. Lorenz, Richard J. O’Reilly, David A. Scheinberg
Tao Dao, Sung Soo Mun, Zaki Molvi, Tatyana Korontsvit, Martin G. Klatt, Abdul G. Khan, Elisabeth K. Nyakatura, Mary Ann Pohl, Thomas E. White, Paul J. Balderes, Ivo C. Lorenz, Richard J. O’Reilly, David A. Scheinberg
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Research Article Immunology Oncology

A TCR mimic monoclonal antibody reactive with the “public” phospho-neoantigen pIRS2/HLA-A*02:01 complex

Abstract

Phosphopeptides derived from dysregulated protein phosphorylation in cancer cells can be processed and presented by MHC class I and class II molecules and, therefore, represent an untapped class of tumor-specific antigens that could be used as widely expressed “public” cancer neoantigens (NeoAgs). We generated a TCR mimic (TCRm) mAb, 6B1, specific for a phosphopeptide derived from insulin receptor substrate 2 (pIRS2) presented by HLA-A*02:01. The pIRS2 epitope’s presentation by HLA-A*02:01 was confirmed by mass spectrometry. The TCRm 6B1 specifically bound to pIRS2/HLA-A2 complex on tumor cell lines that expressed pIRS2 in the context of HLA-A*02:01. Bispecific mAbs engaging CD3 of T cells were able to kill tumor cell lines in a pIRS2- and HLA-A*02:01–restricted manner. Structure modeling shows a prerequisite for an arginine or lysine at the first position to bind mAb. Therefore, 6B1 could recognize phosphopeptides derived from various phosphorylated proteins with similar amino acid compositions. This raised the possibility that a TCRm specific for the pIRS2/HLA-A2 complex could target a range of phosphopeptides presented by HLA-A*02:01 in various tumor cells. This is the first TCRm mAb to our knowledge targeting a phosphopeptide/MHC class I complex; the potential of this class of agents for clinical applications warrants further investigation.

Authors

Tao Dao, Sung Soo Mun, Zaki Molvi, Tatyana Korontsvit, Martin G. Klatt, Abdul G. Khan, Elisabeth K. Nyakatura, Mary Ann Pohl, Thomas E. White, Paul J. Balderes, Ivo C. Lorenz, Richard J. O’Reilly, David A. Scheinberg

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Figure 5

Cytotoxicity by 6B1 BisAbs.

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Cytotoxicity by 6B1 BisAbs. (A–D) T2 cells alone (A) or pulsed with pIRS...
(A–D) T2 cells alone (A) or pulsed with pIRS2 (B), WT-IRS2 (C), or irrelevant EW (D) peptides at 50 μg/mL were incubated with PBMCs at an E: T of 30:1 and mAbs at indicated concentrations, and the cytotoxicity was measured by a 5-hour 51Cr-release assay. Each data point was the average of triplicate cultures ± SD; data are representative of 2 similar experiments. (E–H) Similarly, T cell–mediated cytotoxicity against tumor cell lines MDA-MB-231 (E), SKOV-3 (F), TPC-1 (G), or A375 (H) was measure by LDH-release assay using EBV-T cells at an E:T ratio of 20:1, with BisAbs at the indicated concentrations. Each data point was the average of triplicate cultures ± SD; data are representative of 3 similar experiments. (I and J) AML PDXs were stained with mAbs to CD33 and HLA-A2. The cells were labeled with CFSE and were incubated with activated T cells at an E:T ratio of 6:1, in the presence of 6B1 (1+1) or isotype control at 10 μg/mL. After overnight culture, cells were harvested, washed, and stained with mAb to CD33. Percentage reduction of total CFSE+ cells was determined as killing of the cells. (K) The percentage lysis of 6B1 (1+1) group was plotted over isotype control.