Label monoclonal antibodies site specifically with ETAC reagents

ETAC and labeling monoclonal antibodies

Monoclonal antibodies and their small fragments (Fabs, scFv, diabodies etc.) are intriguing objects for creation of protein-based medicines. These proteins can be site-specifically modified with ETAC-dPEG® (“ETAC” abbreviates “Equilibrium Transfer Alkylation Cross-link”; “dPEG®” is the registered trade name for “discrete Poly(Ethylene Glycol)”) reagents. Using ETAC, a three-carbon bridge is formed linking the two cysteine sulfur atoms. The dPEG® attached to the ETAC reduces the protein’s immunogenicity and prevents rapid clearance of the protein from the bloodstream. This, in turn, helps to maintain a desired therapeutic concentration between doses, thereby reducing the risk of loss of efficacy. The structure of ETAC-reagent and generation of the dPEG®-monosulfone which undergoes a site-specific conjugation with a Fab are outlined below in Figure 1. For details, see, for example, “Comparative binding of disulfide-bridged PEG-Fabs”, Bioconjugate Chemistry (2012), 23, 2262-2277; and “Disulfide bridge based PEGylation of proteins”, Advances in Drug Delivery Reviews (2008), 60, 3-12.

Monoclonal antibodies can be site specifically labeled using the ETAC-dPEG® reagent shown here, after generation of the monosulfone as depicted in the scheme.
Figure 1: Structure of the ETAC-dPEG® reagent and generation of the monosulfone to label monoclonal antibodies site specifically.

An accessible disulfide bond can be selectively reduced under mild conditions with DTT or TCEP (Quanta BioDesign product number PN10014) without destroying the tertiary structure of the monoclonal antibody or antibody fragment. Once an accessible disulfide is reduced, the two free cysteine sulfur atoms become available for reaction with the ETAC-reagent (See “Disulfide bridge based PEGylation of proteins”). The PEG-bis-sulfone eliminates the sulfinic anion and generates PEG-monosulfone which then undergoes conjugation, particularly with Fabs. Conjugation occurs by the formation of a three-carbon bridge linking the two cysteine sulfur atoms with PEG attached through the middle carbon of the bridge. Conjugation of PEG at this site normally has minimal impact on the binding properties of the monoclonal antibody or Fab. Figure 2, below, shows a generalized scheme for this process.

General scheme showing how monoclonal antibodies or other disulfide-containing proteins can be site-specifically labeled using an ETAC-dPEG® reagent.
Figure 2: General scheme showing how monoclonal antibodies or other disulfide-containing proteins can be site-specifically labeled using an ETAC-dPEG® reagent.

Potential problems with PEGylated monoclonal antibodies as drugs

Having high molecular weight dPEG® in solution can stabilize the native and compact structure of human albumin, does not obscure the protein’s active surface, and folds independently of the protein. The majority of clinically used PEGylated medicines are heterogeneous mixtures that have been produced by non-specific and inefficient PEG-conjugation reaction to different nucleophilic sites on the protein ( See “Comparative binding of disulfide-bridged PEG-Fabs”).

Several monoclonal antibodies marketed as drugs use maleimide to conjugate PEG to thiol. Examples of PEGylated monoclonal antibodies manufactured this way include the drug certolizumab pegol (tradename Cimzia) and the now-withdrawn drug peginesatide, a PEGylated peptide.  Although, thiol conjugation is quite efficient, the addition of unpaired cysteine can result in disulfide scrambling and protein aggregation. In addition, there are data that maleimide derived reagents are labile to hydrolysis and can undergo exchange reactions in vivo. See Reversible maleimide-thiol adducts yield glutathione-sensitive poly(ethylene glycol)-heparin hydrogels. Polym Chem (2013), 4, 133-143; and Tunable degradation of maleimide-thiol adducts in reducing environments. Bioconjugate Chem (2011), 22(10), 1946-1953. Therefore, development of site-specific approaches and novel reagents that address low conjugation efficiency and PEG-conjugate stability is important, especially in PEGylated monoclonal antibodies and Fab fragments that have potential to be used as therapeutic agents.

ETAC-dPEG® reagents offer advantages over traditional PEGylation reagents

Our novel heterobifunctional ETAC-dPEG® (linear or branched) reagents with active TFP- or NHS-groups (for example, ETAC-dPEG®24-NHS ester, PN11685, Figure 3, and ETAC-dPEG®36-TFP ester, PN11686, Figure 4) can be used for preparation of homogeneous, highly potent antibody drug conjugates. It allows to combine the unique targeting capabilities of monoclonal antibodies with therapeutic or diagnostic payload (cytotoxic drug, toxin), dPEG®– and cleavable or noncleavable linker. As a result, the targeted cell (e.g. cancerous) can be damaged either by the released cytotoxic drug, or by the complex of degraded antibody, linker, and drug.

Figure 3: PN11685, ETAC-dPEG®-NHS ester, can be used to label monoclonal antibodies or other proteins site specifically. For more information, please contact Quanta BioDesign, Ltd.
Figure 3: PN11685, ETAC-dPEG®24-NHS ester, is a single molecule (not polydispersed) PEGylation reagent that can be used to label monoclonal antibodies or other proteins site specifically. For more information, please contact Quanta BioDesign, Ltd.
Figure 4: PN11686, ETAC-dPEG®36-TFP ester, is a single molecule (not polydispersed) PEGylation reagent that can be used to label monoclonal antibodies site specifically. For more information, please contact Quanta BioDesign, Ltd.
Figure 4: PN11686, ETAC-dPEG®36-TFP ester, is a single molecule (not polydispersed) PEGylation reagent that can be used to label monoclonal antibodies site specifically. For more information, please contact Quanta BioDesign, Ltd.

It is important to note that a dPEG® is a single molecular species. (See “What is dPEG®?“) Traditional PEGylation reagents are dispersed polymer mixtures. Working with a traditional, polydispersed PEG makes complete characterization of the final product (whether small molecules, peptides, monoclonal antibodies, or other proteins) difficult, because the complex heterogeneity of the polydispersed PEG makes it intractable to analysis.

PEGylation reagents for all applications

More than just ETAC reagents, Quanta BioDesign, Ltd., offers PEGylation reagents for almost every need in bioconjugation, therapeutics, diagnostics, theranostics, nanotechnology, bionanotechnology, and many other fields. Our functional groups are diverse, and our chemistry allows us to make a broad range of dPEG® linkers from 1-49 ethylene glycol units as single molecular entities. Look through our catalog today! If you cannot find the linker chemistry you want for your specific application, please feel free to call us and ask. We offer custom synthesis of dPEG® reagents to our customers. We will be happy to speak with you and discuss your needs.  Just call us today!

Links to additional PEGylation reagents of interest

TCEP – an effective reagent for reducing disulfides to thiols.

Biotinylation Reagents – biotinylate almost anything with one of our Biotin-dPEG® reagents.

Crosslinking reagents with a variety of chemistry options, including thiol-reactive maleimide and SPDP.

Fluorescent and dye labels – label monoclonal antibodies or other proteins.

Victor D. Sorokin, Ph.D. – Received his Ph.D. in Organic Chemistry from the Moscow State University in Russia and completed his postdoctoral training at the University of Texas (1993-1996) where he started his industrial career. He has recently joined our company as a Sr. Product Development Scientist. He has over 15 years of synthetic organic chemistry experience with chemical and pharmaceutical companies developing the synthesis of complex molecules (including natural chiral compounds) on mg/g scale, allowing scale-up to multi-kg quantities. For the last 5 years he has been working on the synthesis of various nucleosides (both DNA, RNA) and oligonucleotides in solution phase using H-phosphonate and phosphoramidate chemistry approach. You can contact Victor on LinkedIn at www.linkedin.com/pub/victor-sorokin/11/417/34b.

 

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