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. 2023 Mar 7;24(6):5134.
doi: 10.3390/ijms24065134.

Aminooxy Click Modification of a Periodate-Oxidized Immunoglobulin G: A General Approach to Antibody-Drug Conjugates with Dye-Mediated Expeditious Stoichiometry Control

Ksenia A Sapozhnikova  1 , Evgeny L Gulyak  1 , Vladimir A Brylev  1   2 , Vsevolod A Misyurin  3 , Sergey D Oreshkov  1   4 , Anastasiya V Alexeeva  5 , Dmitry Yu Ryazantsev  1 , Maria A Simonova  1 , Ekaterina V Ryabukhina  1 , Galina P Popova  1 , Nataliya A Tikhonova  5 , Natalia A Lyzhko  5 , Alexander E Barmashov  3 , Andrey V Misyurin  5 , Alexey V Ustinov  1   2 , Vera A Alferova  1 , Vladimir A Korshun  1
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Aminooxy Click Modification of a Periodate-Oxidized Immunoglobulin G: A General Approach to Antibody-Drug Conjugates with Dye-Mediated Expeditious Stoichiometry Control

Ksenia A Sapozhnikova et al. Int J Mol Sci. .

Abstract

A universal approach to the construction of antibody-drug conjugates (ADCs) has been developed. It relies on periodate oxidation of naturally present glycans of immunoglobulin G, followed by oxime ligation and, optionally, copper(I)-catalyzed alkyne-azide cycloaddition for conjugation with a toxic payload. The introduction of highly absorbing cyanine dyes into the linker allows for facile determination of the drug-antibody ratio. We applied this methodology to the synthesis of cytotoxic conjugates of an antibody against the tumor-associated antigen PRAME with doxorubicin and monomethyl auristatin E (MMAE). The resultant conjugates retained their affinity to a large extent, yet their cytotoxicity in vitro varied dramatically: while the doxorubicin-based conjugate did not produce any effect on cells, the MMAE-based one demonstrated specific activity against PRAME-expressing cancer cell lines. Importantly, the latter conjugate constitutes the first reported example of a PRAME-targeting ADC.

Keywords: ADC; CuAAC; MMAE; PRAME; antibody; cleavable linkers; cyanine dyes; doxorubicin; oxime ligation; periodate oxidation.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
The methodology affords a site-specific ADC with facile stoichiometry control.
Scheme 1
Scheme 1
Four approaches (A–D) to the assembly of doxorubicin-based ADCs from periodate-oxidized IgG via oxime ligation with cyanine dyes in the linker.
Scheme 2
Scheme 2
Synthetic scheme for approach A.
Scheme 3
Scheme 3
Side reaction in the course of ADC synthesis via approach A.
Scheme 4
Scheme 4
Model click reaction for drug–antibody ratio determination via approach B.
Figure 2
Figure 2
UV/Vis absorbance spectra of 5 and 18 compared to sCy3-sCy5 conjugate 19 in aqueous solution.
Scheme 5
Scheme 5
Synthesis of reagents for approach C.
Scheme 6
Scheme 6
Synthesis of reagents for approach D.
Figure 3
Figure 3
UV/Vis absorbance spectra of intermediate 13 and ADC 14 in PBS.
Figure 4
Figure 4
Fluorescence spectrum of ADC 14 (emission spectrum, excitation wavelength: 520 nm).
Scheme 7
Scheme 7
Synthesis of reagent 31.
Figure 5
Figure 5
UV/Vis absorbance spectrum of conjugate 32.
Figure 6
Figure 6
Affinity of conjugates 13, 14, and 32 analyzed by ELISA.
Figure 7
Figure 7
Viability of PRAME (+) human melanoma cell line MelP analyzed by MTT assay following 72 h incubation with 31, 32, MMAE, and 6H8 (A); 14, 10, doxorubicin, and 6H8 (B).
Figure 8
Figure 8
Viability of PRAME (+) human acute monocytic leukemia cell line THP-1 analyzed by MTT assay following 72 h incubation with 31, 32, MMAE, and 6H8 mAb (A); 14, 10, doxorubicin, and 6H8 (B).
Figure 9
Figure 9
Viability of PRAME (-) murine colorectal carcinoma cell line CT26 analyzed by MTT assay following 72 h incubation with 31, 32, MMAE, and 6H8 mAb (A); 14, 10, doxorubicin, and 6H8 (B).
Figure 10
Figure 10
The structure of conjugate 5.
Figure 11
Figure 11
The structure of conjugate 9.
Figure 12
Figure 12
The structure of conjugate 13.
Figure 13
Figure 13
The structure of conjugate 3.
Figure 14
Figure 14
The structure of conjugate 32.
Figure 15
Figure 15
The structure of conjugate 18.
Figure 16
Figure 16
The structure of conjugate 11.
Figure 17
Figure 17
The structure of conjugate 14.
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