Intein-fusion Technology Based Conjugation

Taking advantage of the intein-fusion technology, Creative Biolabs provides our clients with high-quality antibody-drug conjugates (ADCs) in a time efficient manner. Intein-fusion technology is developed based on inteins, a class of peptides that undergo self-cleavage when inserted into precursor proteins. Intein-fusion has been widely used to produce recombinant proteins with site-specific incorporation of a diverse set of chemical modifications. Recently, this technology has been used as an innovative solution for specific C-terminal modification of antibodies and further facilitates the site-directed conjugation with various cytotoxic drugs to generate homogeneous ADC products. At Creative Biolabs, we have established two different strategies for intein-fusion, namely expressed protein ligation (EPL) and protein trans-splicing (PTS). We are committed to offering top-quality conjugation service using these techniques to meet your ADC development requirements.

Expressed protein ligation (EPL) strategy

Expressed protein ligation (EPL) is one of the classic approaches that introduce intein-mediated C-terminal protein modifications. GyrA intein from Mycobacterium xenopi (Mxe), also known as “minimal” intein, is one of the representative inteins involving in EPL and it is comprised of 198 amino acid residues. To modify an antibody using EPL approach, a modified full-length GyrA intein is first fused to the C terminus of the targeted antibody through recombinant protein expression. After intein-catalyzed N-S acyl shift, the GyrA intein is cleaved off from the antibody by the addition of a thiol reagent, such as Mesna, resulting in a C-terminus thioester bond. Cys-functionalized drug or drug-linker complexes are then conjugated to the antibody and forming a stable peptide bond with the antibody C-terminus and the subsequent S-N acyl shift generates a new thiol group that forms a disulfide bond with its other Ct counterpart, further stabilizing the Ct conjugation area.

Intein-fusion technology based conjugation Illustration of steps during in vitro processing of IgG-intein fusion protein (top: Ct conjugation via GyrA intein; bottom: conjugation via DnaE intein, BMC Biotechnology, 2011).

Protein trans-splicing (PTS) strategy

Protein trans-splicing, abbreviated as PTS, belongs to intein-mediated protein splicing which turns out to be an essential tool for promoting efficient protein modification, ligation, cyclization, and purification. The split DnaE intein from Nostoc punctiforme (Npu) is one of the commonly used inteins in PTS. DnaE intein is comprised of an N-terminal portion with 102 amino acid residues and a C-terminal portion with 36 amino acid residues. The two sections exhibit high affinity to each other. For antibody modification and conjugation via PTS, the N-terminal portion of DnaE intein is fused to the heavy chain C-terminus of the target antibody, resulting in two inteins per antibody. In the meantime, the C-terminal portion of the DnaE intein is fused to the drug-linker complex. The association of the two parts re-constitutes a functional intein which splices and releases itself from the antibody and concomitantly, fuses the target antibody to the drug-linker complexes.

Advantages of intein-fusion technology base antibody modification and conjugation:

  • Suitable for the modifications on the Fc region of an antibody
  • Generating site-specific ADCs with a drug to antibody ratio (DAR) of 1 or 2
  • Modification and conjugation targets to the C- terminus of an antibody that exert no significant influence on affinity and effector recruiting functions of the antibody

With years of experience in antibody engineering, Creative Biolabs is committed in bringing in new technology and providing 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.

Reference:

  1. Möhlmann, S.; et al. Site-specific modification of ED-B-targeting antibody using intein-fusion technology. BMC Biotechnology. 2011, 11: 76.


Related Sections

One-stop ADC Development Service: Services:
Online Inquiry
Name:
*Phone:
*E-mail Address:
*Products or Services Interested:
Company/Institution
*Project Description:
*Verification Code:
Please input "biolabs"(case insensitive) as verification code.


Welcome! For price inquiries, please feel free to contact us through the form on the left side. We will get back to you as soon as possible.


Contact us
 45-1 Ramsey Road, Shirley, NY 11967, USA
 Tel: 1-631-381-2994
 Fax: 1-631-207-8356
 Email:

Inquiry

Top