Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2018 Jul;10(5):712-719.
doi: 10.1080/19420862.2018.1463122.

N-terminal α-amino group modification of antibodies using a site-selective click chemistry method

De-Zhi Li  1 , Bing-Nan Han  2 , Rui Wei  3 , Gui-Yang Yao  4 , Zhizhen Chen  3 , Jie Liu  3 , Terence C W Poon  3 , Wu Su  4 , Zhongyu Zhu  5 , Dimiter S Dimitrov  6 , Qi Zhao  3
Affiliations

N-terminal α-amino group modification of antibodies using a site-selective click chemistry method

De-Zhi Li et al. MAbs. 2018 Jul.

Abstract

Site-specific conjugation of small molecules to antibody molecules is a promising strategy for generation of antibody-drug conjugates. In this report, we describe the successful synthesis of a novel bifunctional molecule, 6-(azidomethyl)-2-pyridinecarboxyaldehyde (6-AM-2-PCA), which was used for conjugation of small molecules to peptides and antibodies. We demonstrated that 6-AM-2-PCA selectively reacted with N-terminal amino groups of peptides and antibodies. In addition, the azide group of 6-AM-2-PCA enabled copper-free click chemistry coupling with dibenzocyclooctyne-containing reagents. Bifunctional 6-AM-2-PCA mediated site-specific conjugation without requiring genetic engineering of peptides or antibodies. A key advantage of 6-AM-2-PCA as a conjugation reagent is its ability to modify proteins in a single step under physiological conditions that are sufficiently moderate to retain protein function. Therefore, this new click chemistry-based method could be a useful complement to other conjugation methods.

Keywords: Site-specific modification,; antibody; antibody engineering; antibody-drug conjugate; click chemistry.

PubMed Disclaimer

Figures

Figure 1.
Figure 1.
Synthesis of 6-AM-2-PCA and its application for N-terminal modification of peptides or proteins. (A) 6-AM-2-PCA is synthesized through azidization and subsequent oxidation of 6-(bromomethyl)-2-pyridinemethanol (1) to yield an azido intermediate (2) and ultimately, pyridinecarboxyaldehyde (3). (B) N-terminal modification of a protein with 6-AM-2-PCA results in an azide appended to the protein that orthogonally reacts with a DBCO derivative through the aldehyde-amine reaction.
Figure 2.
Figure 2.
Fluorescence imaging of Fab and IgG antibodies modified by 6-AM-2-PCA. (A) Fab antibodies were treated with 6-AM-2-PCA, following by fixing to the membrane and incubation with DBCO-Seta650. Fluorescence signal was detected. (B) IgG antibodies were treated with 6-AM-2-PCA, following by fixing to the membrane and incubation with DBCO-Cy5.5. Fluorescence signal was detected. (C) IgG antibodies were treated with 6-AM-2-PCA, and sequentially labeled with DBCO-Cy5.5. The upper panel is the fluorescent imaging. The lower panel is Coomassie brilliant blue R-250 staining of proteins.
Figure 3.
Figure 3.
Site-specific attachment of 6-AM-2-PCA to ananti-Her2 antibody analyzed by Q Exactive MS. (A) Fab was reacted with small molecules and analyzed by LC/MS. A peak at 50359 Da (1) was assigned to unmodified Fab. The peaks at 50503 Da (2) and 50647 Da (3) are assigned to mono-modified and bis-modified Fab products, respectively, from the completed aldehyde-amine reaction. The peak at 50665 Da (4) represents a hemiaminal product for the aldehyde-amine reaction. Data are representative of two experimental observations. (B) Two mechanisms of the aldehyde-amine reaction.
Figure 4.
Figure 4.
Binding kinetic analysis of naive and modified anti-Her2 IgG antibodies to Her2 antigen. (A) Sensorgrams of binding kinetics. (B) Summary of binding rate constants and KD values. Experimental details are described in Materials and Methods.
Figure 5.
Figure 5.
The stability of modified and unmodified antibodies in mouse plasma
Figure 6.
Figure 6.
Immunostaining of SK-BR-3 breast cancer cells by 6-AM-2-PCA-conjugated anti-Her2 antibodies. (A) Three panels (left to right) represent fluorescence of SK-BR-3 cells stained with modified anti-Her2 Fab followed by DBCO-ATTO 488, nuclei stained with DPAI, and the mergedimage. (B) Three panels (left to right) represent fluorescence of SK-BR-3 cells stained with DBCO-ATTO 488 alone, nuclei stained with DPAI, and the merged image.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Leavy O. Therapeutic antibodies: past, present and future. Nat Rev Immunol. 2010;10:297. doi:10.1038/nri2763. PMID:20422787. - DOI - PubMed
    1. Adler MJ, Dimitrov DS. Therapeutic antibodies against cancer. Hematol Oncol Clin North AMa. 2012;26:447–81. vii. doi:10.1016/j.hoc.2012.02.013. PMID:22520975. - DOI - PMC - PubMed
    1. Bakhtiar R. Antibody drug conjugates. Biotechnol lett. 2016;38:1655–64. doi:10.1007/s10529-016-2160-x. PMID:27334710 DOI. - DOI - PubMed
    1. Borcoman E, Le Tourneau C. Antibody drug conjugates: the future of chemotherapy? Curr Opin Oncol. 2016;28:429–36. doi:10.1097/CCO.0000000000000310. PMID:27366963. - DOI - PubMed
    1. Diamantis N, Banerji U. Antibody-drug conjugates–an emerging class of cancer treatment. Br J Cancer. 2016;114:362–7. doi:10.1038/bjc.2015.435. PMID:26742008. - DOI - PMC - PubMed

Publication types

Grants and funding

Start-up Research Grant of University of Macau, SRG2016-00082-FHS, the Science and Technology Development of Macau, FDCT/131/2016/A3.