Home › Immunology › Genetic Encoding of a Non-Canonical Amino Acid for the Generation of Antibody-Drug Conjugates Through a Fast Bioorthogonal Reaction
Immunology JoVE (Open Access) Citable · DOI

Genetic Encoding of a Non-Canonical Amino Acid for the Generation of Antibody-Drug Conjugates Through a Fast Bioorthogonal Reaction

DOI: 10.3791/58066-v
What you'll learn
  • ✓ Produce and characterize antibodies bearing non-canonical cyclopropene-lysine residues
  • ✓ Perform site-specific bioorthogonal conjugation of tetrazine-bearing payloads to antibodies
  • ✓ Evaluate ADC stability, potency, and selectivity in vitro
Protocol

Incorporating a cyclopropene derivative of lysine into antibodies allows the site-specific, rapid and efficient linkage of tetrazine-bearing molecules to generate antibody-drug conjugates.

Difficulty
advanced
Total time
~3–5 days (antibody production and characterization ~2–3 days; conjugation and assays ~1–2 days)
Model organism
HEK293 (mammalian expression cells)
Biosafety
BSL-1

Steps

1
Produce and characterize engineered antibody

Express antibody containing cyclopropene-modified lysine residues in mammalian cells and confirm expression and purity via standard analytical techniques.

▶ 00:30
2
Conjugate antibody with tetrazine-bearing drug payload

Perform bioorthogonal inverse-electron-demand Diels–Alder reaction between cyclopropene-lysine on antibody and tetrazine-conjugated small-molecule cargo to generate antibody-drug conjugates.

▶ 03:47
3
Assess dihydropyridazine linkage stability in serum

Incubate conjugated antibody in human serum at physiological conditions and monitor linker stability and payload retention over time.

▶ 06:38
4
Evaluate ADC cytotoxicity in target cell lines

Test antibody-drug conjugate potency and selectivity by measuring cell viability and apoptosis in antigen-positive and antigen-negative cell populations.

▶ 07:46
5
Interpret ADC stability and functional performance

Analyze results confirming that the dihydropyridazine-linked ADC is serum-stable, potent against target cells, and selective relative to non-expressing controls.

▶ 09:11
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