Epitope mapping of PD-L1 primary antibodies (28-8, SP142, SP263, E1L3N).

Abstract

3028 Background: Currently, programmed death-ligand 1 (PD-L1) immunohistochemistry (IHC) assays received approval in combination with an anti-PD-1 or anti-PD-L1 compounds. However, the FDA blueprint and other publications revealed differences in staining pattern between PD-L1 IHC assays. More precisely the SP142 assay detects less tumor cells (TC), but more immune cells (IC), while the 28-8 assay is more sensitive for TC and less appropriate for IC detection. SP263 stains TC and IC equally well. E1L3N IHC reveals IC and slightly more TC staining than SP142. This study investigates whether these staining discrepancies can be partly explained by specific epitope recognition. Methods: Linear epitope mapping was performed for PD-L1 antibody clones 28-8, E1L3N, SP142 and SP263. In brief, the PD-L1 sequence (Q9NZQ7, Uniprot) was split into 15 amino acid (AA) peptides with a peptide overlap of 14 AA . Each peptide was printed in duplicate on the PD-L1 microarray. The microarray was exposed to different concentration of the four PD-L1 antibodies. For the detection, sheep anti-rabbit IgG DyLight800 was used. Results: Epitope mapping for the E1L3N antibody revealed a linear epitope in the intracellular domain. The other clones showed weaker binding to multiple peptides. The 28.8 clone demonstrated binding to predominantly intracellular epitopes, while SP142 and SP263 clones showed binding to both intracellularly and extracellularly located epitopes. Blasting the epitope sequences of the PD-L1 antibodies did not disclose identity with another (non-PD-L1) human protein. Conclusions: Different binding characteristics were found for all four PD-L1 antibody clones in a linear epitope mapping experiment. This could lead to particular staining patterns depending on PD-L1 conformation (folding) or isoform expression. Two PD-L1 isoforms are known, with isoform 2 lacking AA 19-132 of the extracellular domain. Especially SP142 binding can be impacted in the case of dominant isoform 2 presence, since epitopes were observed in this spliced domain. Further investigation is needed into the potentially conformational epitopes of SP142, SP263 and 28.8 antibody clones as well as in the PD-L1 conformation and isoform expression in TC and IC.