Unnatural Amino Acids (UAA) Based Conjugation

Creative Biolabs provides unnatural amino acids (UAAs) containing antibodies to facilitate the generation of antibody-drug conjugates (ADCs). The incorporation of unnatural amino acids into antibody sequences has been demonstrated as an efficient approach for site-specific modification of antibodies and subsequently, the production of homogeneous ADCs. At Creative Biolabs, we introduce two strategies for UAA insertions: cell-based amber codon suppression and cell-free amber codon suppression. Moreover, we also offer Selenocysteine incorporation as another method for conjugation-site specific ADC production. We are committed to develop top-quality ADC products to meet your requirements, timeline, and R&D budget.

Cell-based Amber Codon Suppression

This strategy relies on the introduction of corresponding orthogonal tRNA/aminoacyl-tRNA synthetase (aaRS) pair to install the UAAs at the designed sites on an antibody using amber stop codons. This is the most widely used method for generating UAA containing proteins. Incorporating an UAA with a chemically unique side chain at the desired locations adds orthogonal functionality to the antibody. This process requires small alterations to the antibody protein sequences. Generally, an antibody with sequence containing amber stop codon (TAG) at designed sites is expressed in host cells while another vector containing an orthogonal tRNA/aminoacyl-tRNA synthetase pair is co-expressed with the antibody. UAAs are supplied in the culture media and incorporated into the mutant antibody TAG sites. Commonly used UAAs include p-acetophenylalanine (pAcF), p-azidomethyl-L-phenylalanine (pAMF), and azido-lysine (AzK) and their side chains offer novel reactivity for the conjugation of various payloads. What’s more, two different UAAs can be simultaneously inserted into the same antibody, enabling the conjugation of different toxins with distinct cell-killing mechanisms and thereby increase ADC potency. At Creative Biolabs, both Escherichia. coli UAA expression systems and mammalian cell UAA expression systems are available for the site-direct UAA incorporation into the antibody of interest.

Cell-free Amber Codon Suppression

This strategy combines amber suppression with cell-free protein expression systems (open cell-free synthesis or OCFS). Comparing to cell-based amber codon suppression strategy, this approach works without the requirement of intact living cells and the UAA containing antibodies are synthesized by mixing cell extract with chemical substrates, an energy regeneration system, and the corresponding DNA template. The significant advantage of using cell-free amber codon suppression system is that a large number of conditions for protein expression and different sites for the UAA insertion can be quickly screened simultaneously in microtiter plates.

Unnatural amino acids (UAA) based conjugation Cell-based amber codon suppression strategy (left) and cell-free amber codon suppression strategy (OCFS, right, Antibodies, 2015)

Selenocysteine Incorporation

As one type of non-canonical amino acid (NCAA), selenocysteine (Sec) is structurally similar to the classic amino acid, cysteine, but contains a selenium atom instead of sulfur. The selenol group empowers selenocysteine with more nucleophilic properties than thiol and enables specific conjugation with electrophilic moieties, such as maleimide or iodoacetamide.

Naturally, selenocysteine is incorporated into nascent polypeptides in response to the opal stop codon (UGA) when a Sec insertion sequence (SECIS) is present at the 3′ untranslated regions (UTRs) in eukaryotes, archaea, or immediately downstream of the UGA codon in bacteria. SECIS is an RNA stem-loop and it can recruit several transacting factors to re-code the UGA stop codon for Sec insertion. Selenocysteine are usually engineered into the C-terminus of IgG1 Fc region by inserting the UGA codon and SECIS at the 3’ end of its encoding gene. It has been demonstrated that the incorporation of Sec has no significant influence on antibody structure. In the meantime, selenocysteine conjugation does not impact Fc receptor (FcRn or Fcγ receptors…) binding affinities, thereby preserving the antibody immunogenic activity. Therefore, selenocysteine is a novel strategy for ADC development and enables site-specific drug conjugation at the antibody C-terminus.

Unnatural amino acids (UAA) based conjugation Mechanism of Sec insertion in eukaryotes: SECIS binding protein 2 (SBP2) binds to SECIS elements and simultaneously interacts with Sec-specific translation elongation factor (eEFSec). This protein complex recruits SectRNA[Ser] and Sec, facilitating the incorporation of Sec into the nascent, growing polypeptide (Physiol Rev, 2014).

Advantages of UAA and selenocysteine based conjugations:

  • Generates site-specific, homogeneous ADCs with precisely controlled conjugation sites and consistent drug to antibody ratio (DAR)
  • Minimal structural perturbation
  • Allows the conjugation of a wide variety of bio-orthogonal ligands
  • UAA and selenocysteine incorporation offers high flexibility for ADC preparation
  • Suitable for the modification in whole antibodies as well as in antibody fragments such as Fab, scFv, and Fc fragments

Creative Biolabs provides the most comprehensive antibody modification and conjugation strategies to help our clients with various ADC development projects, please contact us for more information and a detailed quote.


  1. Agarwal, P.; et al. Site-specific antibody−drug conjugates: the nexus of bioorthogonal chemistry, protein engineering, and drug development. Bioconjugate Chem. 2015, 26: 176−192.
  2. Dennler, P.; et al. Antibody conjugates: from heterogeneous populations to defined reagents. Antibodies. 2015, 4: 197-224.
  3. Sochaj, A.M.; et al. Current methods for the synthesis of homogeneous antibody–drug conjugates. Biotechnology Advances. 2015, 33: 775–784.
  4. Vyacheslav, M.; et al. Selenoproteins: molecular pathways and physiological roles. Physiol Rev. 2014, 94: 739 –777.

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