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

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.

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.

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.

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.

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 K_D values. Experimental details are described in Materials and Methods.

Figure 5.

The stability of modified and unmodified antibodies in mouse plasma

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.