Tyrosine Based Conjugation

Creative Biolabs is a leader in antibody engineering and antibody-drug conjugate (ADC) development with over a decade of experience. We now offer a sophisticated novel strategy to conjugate toxins to an antibody via the native amino acid tyrosine. With a strong science team expertise in ADC development as well as state-of-the-art instruments, Creative Biolabs is committed to provide clients with first-class ADC development services in a time and cost efficient manner.

To date, most of ADC preparations are performed by drug conjugation to antibodies via their endogenous lysine or cysteine residues. Although lysine and cysteine are the most common amino acids used for bio-conjugation with highly reactive side chains, these conjugation schemes have their limitations. The high abundance of lysine residues on antibody surfaces makes site-specific modification challenging and generates conjugates with a wide drug to antibody ratio (DAR) distribution. Conjugation using cysteine thiol side chains usually requires partial or full reduction of inter-chain disulfide bonds that are essential for antibody structural integrity. Comparing to lysine and cysteine residues, surface accessible tyrosine residues are rare and their phenolic hydroxyl groups are chemically active to serve as conjugation sites. Therefore, antibody tyrosine residues have emerged as attractive sites for various bio-conjugation attempts. Scientists from Creative Biolabs are experienced in generating customized antibody-drug conjugates by utilizing antibody tyrosine residues and we provide two strategies for customary tyrosine-based conjugation: click-like tyrosine ligation strategy and Tub-tag labeling strategy.

Click-like Tyrosine Ligation Strategy

This strategy is mainly based on the efficient click-like reaction in tyrosine ligation. A drug-linker set is functionalized with 4-phenyl-3H-1,2,4-triazole-3,5(4H)-dione (PTAD), a cyclic diazodicarboxamide derivative, and subsequently conjugated to Tyr via a click-like mechanism, generating tyrosine–based conjugates.

Antibody modification based on click-like tyrosine ligation (Antibodies, 2015).

Tub-tag Labeling Strategy

This is a novel strategy for site-specific antibody modifications that introduces Tyr residues or Tyr derivatives via a highly efficient chemo-enzymatic system. This strategy is based on an enzyme, namely tubulin tyrosine ligase (TTL), which naturally recognizes a 14-amino acid motif at the C-terminus of alpha tubulin (termed as Tub-tag) and catalyzes the post-translational attachment of a Tyr or Tyr derivative to the Tub-tag. At Creative Biolabs, Tub-tag can be fused to the C terminus of an antibody using recombinant protein expression while Tyr or some “unnatural” tyrosine derivatives such as 3-N₃-L-tyrosine can be attached to the Tub-tag with the help of TTL. The drug-linker set is subsequently conjugated to the antibody via the chemical reactivity of Tyr or Tyr derivatives to generate site-specific ADCs.

“Unnatural” tyrosine derivatives are ligated to the C terminus of Tub-tag to serve as 2 iorthogonal handles for the site-specific modification of an antibody (Angew. Chem, 2015).

Advantages of tyrosine based conjugations:

• Modification and conjugation based on tyrosine and has no significant effect on the function and structure of antibodies
• Lower occurrence of tyrosine residues on the surface of an antibody lead to ADC products with higher homogeneity comparing to conjugation via lysine or cysteine
• Click-like tyrosine ligation strategy is performed on native residues with no requirement of antibody engineering
• Tub-tag labeling strategy enables a broad range of high-yielding and fast chemo-selective C-terminal protein modifications on antibodies and the generation of site-specific homogeneous ADCs
• Conjugation based on tyrosine shows stability to extremes of pH, temperature, and exposure to human blood plasma, which is significantly more robust than maleimide-type linkages that are commonly employed in bio-conjugations

Creative Biolabs provides comprehensive antibody modification and conjugation strategies. We are dedicated to provide our clients with high-quality services and contribute to your ADC development projects. Please contact us for more information and a detailed quote.

References:

1. Dennler, P.; et al. Antibody conjugates: from heterogeneous populations to defined reagents. Antibodies. 2015, 4: 197-224.
2. Schumacher, D.; et al. Versatile and efficient site-specific protein functionalization by tubulin tyrosine ligase. Angew. Chem. Int. Ed. 2015, 54: 13787 –13791.