Development of an Oncology VHH-ADC Candidate via Site-Specific Non-Canonical Amino Acid Conjugation

Aug 24, 2026 | Case Studies

Challenge

Incorporate a non-canonical amino acid (ncAA) pAzF into a VHH antibody for ADC enhancement, PET771
Scale to 2L to ensure efficient ncAA incorporation
Confirm binding of new VHH-ADC to target GPCR

Evaluated pAzF substitution sites Y64, Y84, Y98 and T99 shown on VHH protein structures

Strategy

Evaluate three suitable incorporation sites
Screen at 96-deep well scale
Confirm titer and pAzF incorporation by LC/MS
Assess feed strategies at 2L
Determine target binding and conjugation efficiency

Binding activity confirmed: relative MFI versus VHH concentration for native VHH and VHH-ADC

Scale up: titer over time post induction for canonical VHH and ncAA VHH, confirming ncAA incorporation

96-well expression by SDS-CGE and titer for Y84, Y98 and T99, with LC/MS expected and observed masses

Results

96-deep well scale screening confirms titers above 1 mg/mL.
Intact-MS confirms 100% incorporation of pAzF at the expected position with no incorporation of native Tyr or Thr observed
Continuous feed of pAzF with media achieves titers of 15 g/L with 100% pAzF incorporation
VHH-ADC binding confirmed to target GPCR by flow cytometry (data not shown)

PET771 Next Steps

Developing chemical conjugation of PET771 to DBCO-activated antineoplastic agent
In vitro cytotoxicity assays and in vivo efficacy studies to be performed

Discuss Your Project

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