1. Could you clarify how “de-N-glycosylation efficiency” was quantified in Fig. 6e–f? Specifically, how were glycoforms with <4 N-glycans distinguished from fully glycosylated forms on immunoblots, and was this classification validated with complementary techniques?
2. In Fig. 3j–l, you report a ~16-fold reduction in PD-1 binding to PD-L1 after treatment with nbhPDL1-PNGF. How was this fold change calculated from flow cytometry data, and was the binding inhibition confirmed by a quantitative biophysical method such as SPR or ITC? Additionally, how do you interpret the remaining PD-1 binding despite near-complete deglycosylation?
3. Although nbmPDL1-PNGF accumulates in non-tumor tissues such as liver and kidney (Fig. 6), you report little to no off-target deglycosylation. Could you elaborate on how you distinguished protein accumulation from enzymatic activity, and whether any assessment was done for potential deglycosylation of other endogenous glycoproteins in those organs?