Unnatural Amino Acids in ADCs
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Unnatural Amino Acids in ADCs

ADC drug conjugation refers to the method of connecting various components of ADC, which determines the drug-antibody-ratio (DAR) and homogeneity, affecting the drug's activity, stability, and tolerance. It is generally categorized into random conjugation and site-specific conjugation. Common methods for nonspecific conjugation include coupling of lysine residues or cysteine residues. Site-specific conjugation is a developing trend in conjugation methods because it allows for easier control of DAR values and results in more homogeneous ADC drugs. Site-specific conjugation on engineered antibodies can more effectively control DAR and avoid altering antigen binding affinity. Engineered unnatural amino acid site-specific bioconjugation is the addition of unnatural amino acids at specific positions to produce homogeneous products with excellent pharmacokinetics and pharmacodynamics.

Natural Amino Acid Conjugation

Traditional ADCs utilize the amino groups of antibody lysine or the thiol groups of cysteine generated by opening interchain disulfide bonds for conjugation. The amino group of lysine forms an amide bond with an activated carboxylic acid linker, and the thiol group of cysteine reacts with a maleimide moiety. An antibody molecule contains about 80 to 90 lysine residues, and conjugation can occur on nearly 40 different lysine residues. Opening interchain disulfide bonds generates multiple cysteine residues, disrupting the integrity of the antibody molecule. As a result, traditional ADCs are highly heterogeneous, with poor homogeneity (DAR of 1-8), low stability, affecting efficacy and therapeutic window.

Nonspecific conjugationFig. 1 Nonspecific conjugation. (Tsuchikama, 2018)

Site-specific conjugation technology allows for precise and quantitative conjugation of antibodies to small molecule toxins. ADCs produced using this technology have the appropriate DAR, high homogeneity, good stability, high batch-to-batch reproducibility, improved activity, pharmacokinetic properties, and are more suitable for large-scale production.

Engineered Unnatural Amino Acids

Natural amino acids that can be used for conjugation are limited to lysine and cysteine. Unnatural amino acids can be incorporated into recombinant proteins, providing a new avenue for ADC development. In prokaryotic and eukaryotic cells, strategies for introducing non-canonical amino acids (ncAAs) into proteins provide a relatively straightforward approach to site-specific conjugation.

In the process of protein synthesis, various functional entities, including DNA, mRNA, tRNA, amino acids, and aminoacyl-tRNA synthetases (aaRSs) collectively participate. Under the action of aaRSs, each tRNA binds to its corresponding amino acid to form an aminoacyl-tRNA. Subsequently, through its anticodon, it complements the codon on mRNA, attaching the corresponding amino acid to the growing polypeptide chain.

By introducing exogenous tRNAs capable of specifically recognizing unnatural amino acids and their corresponding aaRSs into cells or bacteria, they catalyze the binding of specific amino acids to tRNAs. Upon entering the ribosome, they recognize the amber stop codon (UAG) on mRNA, facilitating site-specific insertion.

During the process of inserting unnatural amino acids, tRNA, amino acids, and aaRSs are mutually exclusive and orthogonally paired. In other words, one type of tRNA can only bind with one type of amino acid, and an aaRS can only bind with one type of aminoacyl-tRNA. Otherwise, the precise information transfer from DNA to mRNA and then to protein cannot be achieved, making it impossible to synthesize the required protein. This unnatural amino acid orthogonal translation technology is also known as the genetic code expansion technique, which utilizes stop codons to insert unnatural amino acids into the amino acid sequence of a protein during the process of translation.

Inserting unnatural amino acids into proteinFig. 2 Inserting unnatural amino acids into protein. (Shandell, 2021)

Non-Canonical Amino Acid Conjugation

The addition of non-canonical amino acids (ncAAs) offers a new possibility for site-specific conjugation. This technology uses amino acids with unique chemical structures, enabling the introduction of linker-drug complexes in a chemically selective manner. It requires the recombination of antibody sequences and the use of tRNAs and aaRSs that are orthogonal to all endogenous tRNAs and synthetases in host cells to respond to unassigned codons for inserting ncAAs into proteins. Typically, ncAAs are added to the culture medium during fermentation. The selection of unnatural amino acids is critical, as they may trigger immunogenicity. Commonly used ncAAs are analogs of natural amino acids with unique functional groups, such as ketones, azides, cyclopropenes, or dienes. These ncAAs carry ketone or azide groups that can chemically react with drug linkers, producing ADCs with uniform DAR values. Ketones can form oxime bonds with hydroxylamine groups, azides can undergo a copper-catalyzed 1,2,3-triazole cycloaddition reaction with alkynes, and azides can also bind to cyclooctyne without copper catalysis, undergoing an azide-alkyne cycloaddition reaction.

Unnatural amino acid site-specific conjugationFig. 3 Unnatural amino acid site-specific conjugation. (Tsuchikama, 2018)

One bioorthogonal ncAA, para-acetylphenylalanine (pAcF), was initially used for exploring site-specific conjugation. Studies have successfully integrated pAcF into an anti-CXCR4 antibody. The payload Auristatin was efficiently conjugated to the antibody through an oxime bond, resulting in a chemically homogeneous ADC with a DAR value of 2. This ADC demonstrated good in vitro activity and complete clearance of lung tumors in mice.

Due to the acidic conditions required for oxime bonding and the slow release kinetics of ADCs, another option is to introduce azido derivatives of ncAAs. The widely used para-azidophenylalanine (pAzF) can rapidly undergo CuAAC or SPAAC reactions under physiological conditions, successfully conjugating glucocorticoid payloads to an anti-CD74 antibody. Besides the pAcF technology, azido-lysine (AzK) analogs have been successfully incorporated into antibodies to create site-specific ADCs with payloads like Auristatin, PBD dimers, or tubulin inhibitors.

Additionally, N-cyclopropene-L-lysine (CypK), a derivatives of lysine, and naturally occurring ncAAs such as selenocysteine (Sec) have been successfully integrated into antibodies. The resulting ADCs show good stability, selectivity, in vitro and in vivo activity.


  1. Tsuchikama, K., and An, Z., Antibody-drug conjugates: recent advances in conjugation and linker chemistries, Protein Cell., 2018, 9(1): 33-46.
  2. Shandell, M. A., et al., Genetic Code Expansion: A Brief History and Perspective, J. Am. Chem. Soc., Biochemistry, 2021, 60, 46, 3455-3469.
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