A Kinetically Controlled Bioconjugation Method for the Synthesis of Radioimmunoconjugates and the Development of a Domain Mapping MS-Workflow for Its Characterization

Abstract

Immunoconjugates exploit the high affinity of monoclonal antibodies for a recognized antigen to selectively deliver a cytotoxic payload, such as drugs or radioactive nuclides, at the site of disease. Despite numerous techniques have been recently developed for site-selective bioconjugations of protein structures, reaction of ε-amine group of lysine residues with electrophilic reactants, such as activated esters (NHS), is the main method reported in the literature as it maintains proteins in their native conformation. Since antibodies hold a high number of lysine residues, a heterogeneous mixture of conjugates will be generated, which can result in decreased target affinity. Here, we report an intradomain regioselective bioconjugation between the monoclonal antibody Trastuzumab and the N-hydroxysuccinimide ester of the chelator 2,2',2″,2‴-(1,4,7,10-tetraazacyclododecane-1,4,7,10-tetrayl)tetraacetic acid (DOTA) by a kinetically controlled reaction adding substoichiometric quantities of the activated ester to the mAb working at slightly basic pH. Liquid chromatography-mass spectrometry (LC-MS) analyses were carried out to assess the chelator-antibody ratio (CAR) and the number of chelating moieties linked to the mAb chains. Proteolysis experiments showed four lysine residues mainly involved in bioconjugation (K188 for the light chain and K30, K293, and K417 for the heavy chain), each of which was located in a different domain. Since the displayed intradomain regioselectivity, a domain mapping MS-workflow, based on a selective domain denaturation, was developed to quantify the percentage of chelator linked to each mAb domain. The resulting immunoconjugate mixture showed an average CAR of 0.9. About a third of the heavy chains were found as monoconjugated, whereas conjugation of the chelator in the light chain was negligible. Domain mapping showed the CH3 domain bearing 13% of conjugated DOTA, followed by CH2 and VH respectively bearing 12.5 and 11% of bonded chelator. Bioconjugation was not found in the CH1 domain, whereas for the light chain, only the CL domain was conjugated (6%). Data analysis based on LC-MS quantification of different analytical levels (intact, reduced chains, and domains) provided the immunoconjugate formulation. A mixture of immunoconjugates restricted to 15 species was obtained, and the percentage of each one within the mixture was calculated. In particular, species bearing 1 DOTA with a relative abundance ranging from 4 to 20-fold, in comparison to species bearing 2DOTA, were observed. Pairing of bioconjugation under kinetic control with the developed domain mapping MS-workflow could raise the standard of chemical quality for immunoconjugates obtained with commercially available reactants.

Figure 1

Deconvoluted electrospray ionization mass spectrometry (ESI-MS) spectra of deglycosilated immunoconjugates (KC up and one-step synthesized down): intact mass analysis (on the left), deglycosilated heavy chain (in the middle), and light chain (on the right). T = Trastuzumab.

Figure 2

Comparison of experimental (top) and theoretical (bottom) spectra of DOTA-conjugated fragments: (A) m/z 638.81 (charge state +2) corresponding to the fragment [184–190] + DOTA (light chain); (B) m/z 875.43 (charge state +3) corresponding to the carbamidomethylated (CAM) fragment [20–38] + DOTA (heavy chain); (C) m/z 444.24 (charge state +2) corresponding to the fragment [292–295] + DOTA (heavy chain); (D) m/z 602.83 (charge state +2) corresponding to the fragment [413–419] + DOTA (heavy chain).

Figure 3

Schematic representation of the limited proteolysis protocol. Immunoconjugate, denatured at 68 °C during reduction and alkylation steps, followed three main pathways of unfolding. Pathway A: the only CH2 domain is denatured and fully digested, undigested Fab_HC, CH3 domain fragments, and intact LC are generated; pathway B: CH2, VH, and CL domains are denatured and fully digested, generating CH3, CH1, and VL single-domain fragments; pathway C: the entire immunoconjugate is denatured and fully digested. Picture created with BioRender.com.

Figure 4

Domain mapping of the immunoconjugate synthesized under kinetic control.

Figure 5

Deconvoluted spectra of peaks eluting from 25 min onward: (A) fragment [1–108] of the light chain corresponding to the VL domain, no presence of DOTA-conjugated species was revealed; (B) fragment [1–225] carbamidomethylated (CAM) at C223 of the heavy chain corresponding to domain Fab_HC, 11% of its DOTA-conjugated was revealed; (C) oxidized fragment [137–213] of the heavy chain corresponding to the CH1 domain, no presence of DOTA-conjugated species was revealed; (D) fragment [344–442] of the heavy chain corresponding to domain CH3, 13% of its DOTA-conjugated was revealed.

Figure 6

Representation of mixture composition: blue color indicates the most intense species, white color the less. Created with BioRender.com.