In particular, PEG linker constructs are employed to increase the hydrophilicity of the desired final constructs. As payloads are often rather hydrophobic, such PEG linkers help to solubilize the conjugate. As an example, in 2020, Simmon et al. studied a family of MMAE-based ADCs with linkers containing PEG chains of 0, 4, 8, or 12 units. The results revealed that mice showed better dosage tolerance for ADCs with higher hydrophilicity gained by PEG incorporation.

For introducing a PEG moiety into a desired construct, amino-PEG-acids are useful building blocks. They are available with all conventional types of protecting groups and can be incorporated at any sequence position by standard coupling protocols. As we are constantly striving to improve our products and offer solutions for all possible kinds of applications, we are herein presenting our findings on possible side-reactions of certain linker constructs as shown in the figure below.

Possible side-reaction of unmethylated amino-PEG-acid.

To avoid this cyclized side-product which is hampering further conjugation and/or lowering overall yields, Iris Biotech is offering N-methylated amino-PEG-acids (see related products), which can easily be introduced by standard coupling procedures.

As an example, Zimmerman et al. reported on Trastuzumab variants conjugated to an exemplary cytotoxic agent, MMAF, using a constrained cyclooctyne reagent. As one part of the construct, they are using such a N-methylated amino-PEG-acid for hydrophilicity adjustment.

Structure of DBCO-PEG(4)-MMAF bearing a N-methylated amino-PEG-acid as solubilizing unit.

References:

Production of Site-Specific Antibody-Drug Conjugates Using Optimized Non-Natural Amino Acids in a Cell-Free Expression System; E. S. Zimmerman, T. H. Heibeck, A. Gill, X. Li, C. J. Murray, M. R. Madlansacay, C. Tran, N. T. Uter, G. Yin, P. J. Rivers, A. Y. Yam, W. D. Wang, A. R. Steiner, S. U. Bajad, K. Penta, W. Yang, T. J. Hallam, C. D. Thanos, A. K. Sato; Bioconjugate Chem. 2014; 25(2): 351-361. https://doi.org/10.1021/bc400490z.

RP-HPLC DAR Characterization of Site-Specific Antibody Drug Conjugates Produced in a Cell-Free Expression System; Y. Xu, G. Jiang, C. Tran, X. Li, T. H. Heibeck, M. R. Masikat, Q. Cai, A. R. Steiner, A. K. Sato, T. J. Hallam, G. Yin; Org. Process Res. Dev. 2016; 20(6): 1034-1043. https://doi.org/10.1021/acs.oprd.6b00072.

Anti-CD74 antibodies comprising a non-natural amino acid, antibody drug conjugate compositions and therapeutic uses thereof; R. Stafford, A. Yam, A. Gill, K. Penta, X. Li, A. Sato; WO2017132615A1.

Reducing the antigen-independent toxicity of antibody-drug conjugates by minimizing their non-specific clearance through PEGylation; J. K. Simmons, P. J. Burke, J. H. Cochran, P. G. Pittman, R. P. Lyon; Toxicol. Appl. Pharmacol.2020; 392: 114932. https://doi.org/10.1016/j.taap.2020.114932.