A TCR mimic monoclonal antibody for the HPV-16 E7-epitope p11-19/HLA-A*02:01 complex

Abstract

More effective treatments are needed for human papilloma virus (HPV)-induced cancers despite HPV virus vaccination. The oncogenic HPV protein targets are currently undruggable and intracellular and therefore there are no antibodies to these targets. Here we report the discovery of TCR mimic monoclonal antibodies (TCRm mAb) specific for the HPV E7 protein p11-19, YMLDLQPET, when presented on the cell surface in the context of HLA-A*02:01 by use of human phage display libraries. One of the mAbs, 3F8, was able to specifically mediate T cell- redirected cytotoxicity, in a bispecific T cell engager (BiTE) form. While further studies are required to assess the therapeutic potential of this approach, the study provided the proof of concept that TCRm mAb could be a therapeutic strategy for HPV-induced human cancers.

Fig 1. Binding of the mAbs to the HPV-E7p11-19/HLA-A2 complex.

[A] Binding of mAbs to T2 cells pulsed with or without peptides. HPV-E7p11, HPV-E7AAAA or, WT1-RMF peptide at a concentration of 20ug/ml was pulsed onto T2 cells overnight in serum-free RPMI1640 complete medium. Cells were washed and stained with the mAbs 1B1, 2A5 or 3F8 conjugated to APC at a concentration of 3ug/ml. A control TCRm mAb specific for WT1-RMF/HLA-A2 complex, ESK1, was used as a negative control for HPV mAb, but positive assay control for RMF/HLA-A2 complex. In parallel, HLA-A2 expression stabilization was determined by staining the cells with anti-HLA-A2 mAb BB7 clone [B]. Binding potency of the mAbs was measured by titrating the HPV-E7p11-19 peptide at the indicated concentrations onto T2 cells; the cells were stained with indicated mAbs at 3ug/ml [C]. Mab titration was performed for relative avidity on T2 cells pulsed with HPV-E7p11-19 peptide at 20ug/ml and stained with the indicated mAbs at concentrations ranging from 3ug/ml to 0.03ug/ml] [D]. All binding was determined by flow cytometry and indicated by median fluorescence intensity [MFI].

Fig 2. Epitope specificity.

The target HPV-E7p11-19 peptide sequence or the same sequence substituted with alanine at positions 1, 2, 3, 4, 5, 6, 7, 8, 9 indicated as A1 to A9, respectively, and pulsed onto T2 cells at 20ug/ml. The HPV sequence with the 4 middle amino acids substituted with alanine [HPV-E7AAAA] as used to gage binding to the center of the sequence as well. The binding of the mAbs 3F8 [A], 1B1 [B], or 2A5 [C] at a concentration of 3ug/ml was determined by flow cytometric analysis. T2 cells alone, or pulsed with RMF irrelevant peptide were the negative controls. 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 [D]. The data represent one of two similar experiments. [E]. Mirror plot of synthetic [bottom] and cell-derived experimental [top] YMLDLQPET peptides. Relative abundance refers to peak areas which are normalized to maximum peak.

Fig 3. Correlation of HPV mAb binding and HLA-A2 expression.

A panel of cell lines with variable amounts of HLA-A2 on the cell surface were stained with mAbs 1B1, 2A5 and 3F8 at 3ug/ml and BB7.2 to HLA-A2. HPV mAb binding is shown in Y-axis and HLA-A2 expression in X-Axis. [A] A comparison of all 3 mAb on the same plot. Each mAb alone is plotted: 2A5 [B], 1B1 [C], 3F8 [D]. The data is one representative from five independent experiments.

Fig 4. Specificity analysis of the 3F8 BiTE.

Binding of 3F8 to HPV-E7p11/HLA-A2 complex. T2 cells alone [A] or pulsed with HPV-E7p11-19 [B], or RMF peptide [C] at a concentration of 20ug/ml overnight. Cells were washed and stained with 3F8 BiTE [3ug/ml], followed by secondary antibody to His-tag. Binding of 3F8 BiTE to cell surface CD3. Jurkat cells [D] or primary human T cells [E] were stained with 3F8 or control BiTE at a concentration of 3ug/ml, followed by anti-His-PE secondary antibody. A control anti-CD3 antibody was used to confirm the CD3 expression.

Fig 5. Cytotoxicity of the 3F8 BiTE.

T2 cells alone, or pulsed with HPV-E7p11-19 or RMF control peptide at 50ug/ml were incubated with human PBMCs at E:T ratio of 20:1 and the 3F8 BiTE [Fig 5A], or control BiTE [Fig 5B] at indicated concentrations for 5 hrs and the cytotoxicity was measured by ⁵¹Cr-release assay. Similarly, 3F8 BiTE was tested for its cytotoxicity against CasKi [Fig 5C] without HPV E7-11 peptide pulsing at indicated concentrations. Each data point is the average of triplicate cultures and representative of three similar experiments.