Impact of Decalcification, Cold Ischemia, and Deglycosylation on Performance of Programmed Cell Death Ligand-1 Antibodies With Different Binding Epitopes: Comparison of 7 Clones

Abstract

Programmed cell death ligand-1 (PD-L1) expression levels in patients' tumors have demonstrated clinical utility across many cancer types and are used to determine treatment eligibility. Several independently developed PD-L1 immunohistochemical (IHC) predictive assays are commercially available and have demonstrated different levels of staining between assays, generating interest in understanding the similarities and differences between assays. Previously, we identified epitopes in the internal and external domains of PD-L1, bound by antibodies in routine clinical use (SP263, SP142, 22C3, and 28-8). Variance in performance of assays utilizing these antibodies, observed following exposure to preanalytical factors such as decalcification, cold ischemia, and duration of fixation, encouraged additional investigation of antibody-binding sites, to understand whether binding site structures/conformations contribute to differential PD-L1 IHC assay staining. We proceeded to further investigate the epitopes on PD-L1 bound by these antibodies, alongside the major clones utilized in laboratory-developed tests (E1L3N, QR1, and 73-10). Characterization of QR1 and 73-10 clones demonstrated that both bind the PD-L1 C-terminal internal domain, similar to SP263/SP142. Our results also demonstrate that under suboptimal decalcification or fixation conditions, the performance of internal domain antibodies is less detrimentally affected than that of external domain antibodies 22C3/28-8. Furthermore, we show that the binding sites of external domain antibodies are susceptible to deglycosylation and conformational structural changes, which directly result in IHC staining reduction or loss. The binding sites of internal domain antibodies were unaffected by deglycosylation or conformational structural change. This study demonstrates that the location and conformation of binding sites, recognized by antibodies employed in PD-L1 diagnostic assays, differ significantly and exhibit differing degrees of robustness. These findings should reinforce the need for vigilance when performing clinical testing with different PD-L1 IHC assays, particularly in the control of cold ischemia and the selection of fixation and decalcification conditions.

Keywords: PD-L1; cold ischemia; companion diagnostic; decalcification; deglycosylation; epitope mapping; fixation; immunohistochemistry.