A TCR mimic monoclonal antibody reactive with the “public” phospho-neoantigen pIRS2/HLA-A*02:01 complex
Tao Dao, … , Richard J. O’Reilly, David A. Scheinberg
Tao Dao, … , Richard J. O’Reilly, David A. Scheinberg
Published March 8, 2022
Citation Information: JCI Insight. 2022;7(5):e151624. https://doi.org/10.1172/jci.insight.151624.
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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 1

Binding of the 6B1 mAb and epitope specificity.

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Binding of the 6B1 mAb and epitope specificity. (A) Binding of 6B1 to T2...
(A) Binding of 6B1 to T2 cells pulsed with or without peptides. pIRS2, WT-IRS2, pCDC25b, or WT1-RMF peptide at a concentration of 20 μg/mL was pulsed onto T2 cells overnight. Cells were washed and stained with 6B1 mAb at the concentration of 3 μg/mL, followed by secondary mAb staining. The staining included secondary mAb or isotype control human IgG1. (B) In parallel, HLA-A2 expression was determined by staining the cells with anti–HLA-A2 mAb BB7 clone T2. Uns, unstained. (C) 6B1 titration was performed on T2 cells pulsed with indicated peptides and stained with indirect staining with 6B1 at concentrations ranging from 10 μg/mL to 0.1 μg/mL. (D) Binding kinetics of 6B1 was measured by biolayer interferometry (BLI) as indicated in Methods. The pIRS2 peptide sequence was substituted with alanine at positions 1, 2, 4, 5, 6, 7, 8, or 9 or with glycine (G3) indicated as A1–A9, and G3 and the binding of 6B1 (3 μg/mL) was determined by indirect staining and flow cytometric analysis. (E) T2 cells alone or pulsed with HPV peptide were the negative controls. (F) The same cells were simultaneously stained with anti–HLA-A2 mAb, clone BB7.2, to measure the relative binding of the peptides to HLA-A2 molecule. (G) Similarly, threonine substituted peptide with (pT-4) or without phosphate (WT-T4) at the position 4 was pulsed onto T2 cells and the binding of 6B1 mAb was determined by flow cytometry. (H) The same cells were simultaneously stained with anti–HLA-A2 mAb, clone BB7.2, to measure the relative binding of the peptides to HLA-A2 molecule.