UV Photo-crosslinking

With rich experience in the field of antibody engineering and bio-conjugation, Creative Biolabs now offers an innovative strategy for antibody-drug conjugate (ADC) preparation using UV photo-crosslinking. This strategy includes two approaches: (1) UV triggered conjugation onto antibody nucleotide binding sites (NBS) and (2) conjugation via photoactive protein Z. UV-NBS conjugation utilizes UV-energy to initiate a photo-crosslinking reaction between the NBS and the NBS-ligand to achieve controlled site-specific drug to antibody conjugation while the protein Z strategy couples a cytotoxic drug to a photoactive protein Z and then specifically attaches the complex to the Fc region of an antibody upon the exposure to long wavelength UV, generating homogeneous ADCs.

UV-NBS Strategy

The nucleotide binding site (NBS) is an under-utilized, but highly conserved structural site found between the variable light and heavy chains at the Fab domains of nearly every antibody isotype. The NBS is comprised of four amino acid residues with aromatic side chains: Y/F42 and Y/F103 residues in the variable region of the light chain, and Y103 and W118 in the variable region of the heavy chain. When exposed to UV light (254nm), the four conserved residues within NBS form reactive radicals that covalently cross link other aromatic moieties. Therefore, the NBS can service as a useful site for selective conjugation of antibodies to small molecule ligands that contain aromatic rings, such as indole-3-butryicacid (IBA). With a full understanding of UV-NBS mechanism, scientists from Creative Biolabs offer UV-NBS technique to help customers develop site-direct antibody-drug conjugates by taking advantage of peptide linkers and payloads featuring IBA moiety.

UV-NBS Conjugation Procedure

A two-step conjugation strategy can be implemented to facilitate the conjugation of small molecules to an antibody using UV-NBS. The first step is to generate an IBA conjugated version of cysteine (IBA-Thiol) that can be photo-crosslinked to antibodies at the NBS. Ligands that possess either a thiol or a maleimide moiety are subsequently conjugated onto the antibody via the IBA-Thiol. Although IBA- and thiol- active group are both UV sensitive, it has been proved that after UV exposure, the thiol moiety attached to IBA remain active to ensure the highly efficient photo-crosslinking.

UV photo-crosslinking Two-step conjugation strategy of UV-NBS, including the synthesis and UV-induced attachment of IBA-Thiol to the antibody NBS site and the subsequent conjugation with thiol-reactive payloads (Bioconjugate Chem, 2014).

Photoactive protein Z strategy

Native protein Z is a small, helical protein derived from the IgG-binding B domain of Protein A. Protein Z is able to bind most of IgG isotypes and it exerts high binding affinity towards the CH2–CH3 region of the Fc domain. By taking advantage of protein engineering approaches such as unnatural amino acid incorporation and intein mediated expressed protein ligation (EPL), scientists from Creative Biolabs have generated an engineered variant of photoactive protein Z with unnatural amino acid benzoylphenylalanine (BPA) in its Fc-binding domain and several other functional moieties at its C-terminus. BPA serves as a UV-activated cross-linker to attach the photoactive protein Z constructs onto IgG upon the exposure to long wavelength UV (365 nm), while the functional moieties at C-terminus allows the conjugation with various cytotoxic drugs to the antibody. Photoactive protein Z strategy provides a fast and effective approach for the site-specific modification of full length IgG as well as the generation of site-specific ADCs.

UV photo-crosslinking Photoactive protein Z is conjugated specifically to the Fc region of the antibody upon exposure to long wavelength UV (Biotechnol. Adv., 2015).

Advantages of UV-induced photo-crosslinking:

  • Generates site-specific, homogeneous ADCs
  • A simple and reproducible process without the requirement for genetic engineering or pre-activated scaffolds
  • Contains all the benefits of photo-crosslinking
  • High crosslinking efficiency
  • Conjugation is achieved without compromising the antigen binding affinity
  • UV-NBS is suitable for the conjugation to both Fab regions and full length IgG, while photoactive protein Z strategy is compatible with nearly all native form of IgG

Creative Biolabs is committed in exploiting new technologies and providing comprehensive antibody modification and conjugation services to help our clients with various ADC development projects, please contact us for more information and a detailed quote.


  1. Alves, N.J.; et al. Conjugation of a reactive thiol at the nucleotide binding site (NBS) for site-specific antibody functionalization. Bioconjugate Chem. 2014, 25 (7): 1198–1202.
  2. Sochaj, A.M.; et al. Current methods for the synthesis of homogeneous antibody–drug conjugates. Biotechnol. Adv. 2015, 33: 775–784.
  3. Hui, J.Z.; et al. Optimization of photoactive protein Z for fast and efficient site-specific conjugation of native IgG. Bioconjugate Chem. 2014, 25: 1709−1719.

For lab research use only, not for any in vivo human use.

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