A disruptive clickable antibody design for the generation of antibody-drug conjugates

Abstract

Background: Antibody-drug conjugates are cancer therapeutics that combine specificity and toxicity. A highly cytotoxic drug is covalently attached to an antibody that directs it to cancer cells. The conjugation of the drug-linker to the antibody is a key point in research and development as well as in industrial production. The consensus is to conjugate the drug to a surface-exposed part of the antibody to ensure maximum conjugation efficiency. However, the hydrophobic nature of the majority of drugs used in antibody-drug conjugates leads to an increased hydrophobicity of the generated antibody-drug conjugates, resulting in higher liver clearance and decreased stability.

Methods: In contrast, we describe a non-conventional approach in which the drug is conjugated in a buried part of the antibody. To achieve this, a ready-to-click antibody design was created in which an azido-based non-canonical amino acid is introduced within the Fab cavity during antibody synthesis using nonsense suppression technology. The Fab cavity was preferred over the Fc cavity to circumvent issues related to cleavage of the IgG1 lower hinge region in the tumor microenvironment.

Results: This antibody design significantly increased the hydrophilicity of the generated antibody-drug conjugates compared to the current best-in-class designs based on non-canonical amino acids, while conjugation efficiency and functionality were maintained. The robustness of this native shielding effect and the versatility of this approach were also investigated.

Conclusions: This pioneer design may become a starting point for the improvement of antibody-drug conjugates and an option to consider for protecting drugs and linkers from unspecific interactions.

Keywords: antibody-drug conjugates; fab cavity; hydrophilicity; hydrophobicity; native shielding; non-canonical amino acids.

Figure 1

Antibody design. (a) 3D (PDB: 1HZH) and schematic representations of a monoclonal antibody, and schematic representation of an antibody-drug conjugate with the drug within the Fab cavity. (b) 3D representation (PDB: 1HZH) of one of the two Fab regions. Each point represents the α-carbon of the amino acid. The amino groups, the carboxy groups, and the side chains are not represented to simplify the visualization. The sphere represents the α-carbon of the glutamic acid HC-152. (c) Schematic representations of the native antibody sequence with the sphere representing the α-carbon of the glutamic acid HC-152, the antibody sequence with AECK introduced at the position HC-152, and the antibody sequence with AECK at the position HC-152 conjugated to a DBCO-based drug-linker. Only one of the two triazole regioisomers [1, 4] is represented. Amino acids are numbered according to the IgG1-Eu described by Edelman et al. [19].

Figure 2

Antibody synthesis. (a) Schematic representations of the mAb designs A, B, and C, with their intended drug location. (b) Amino acid context, codon context, theoretical iPASS score, and antibody yields in CHO and HEK systems for designs A, B, and C with mAb1 and mAb2. The reference amino acid or reference codon is indicated as underlined. X represents the non-canonical amino acid. iPASS scores were calculated based on the amber codon context by using the website shiny.bio.lmu.de:3838/iPASSv2. An iPASS score of ≥1 should indicate above-average relative non-canonical amino acid incorporation efficiency in HEK cells with the orthogonal M. mazei pyrrolysine-tRNA/pyrrolysine-RS pair. Antibody yields were quantified from the culture supernatants by affinity chromatography using the MabSelect™ SuRe™ column in an ÄKTA pure 25 L system. Transient transfections were independently performed three times. Amino acids are numbered according to IgG1-Eu described by Edelman et al. [19].

Figure 3

ADC hydrophobicity . Left: HIC chromatograms (214 nm) of ADCs based on mAb1 or mAb2, with designs C, A, and B (from top to bottom), and containing SLAE (DBCO-C4-PEG3-VC-PABC-AE). The arrows indicate the corresponding DAR species in each sample. For the chromatograms presented here, 125 ng of mAb1 wild-type (wt) or mAb2 wt were added to the sample (5 μg) as internal standard before injection. Right: schematic representations of ADCs based on designs C, A, and B (from top to bottom). The arrow indicates the increasing hydrophobicity. SLAE stands for standard linker auristatin E and is schematically represented in Figure 4a.

Figure 4

Shielding effect . (a) Chemical structures of the standard linker (SL), long linker (LL), and auristatin E derivatives (AE). SLAE is the combination of SL and AE, in which X is a methyl substituent. LLAE is the combination of LL and AE, in which X is a hydrogen substituent. The dashes at the C-terminus of the linker and at the N-terminus of the drug represent the bond between the linker and the drug. (b) Differences in retention time (RT) between ADCs based on LLAE and ADCs based on SLAE were calculated as follows: RT difference = RT_{LLAE-based ADC}—RT_{SLAE-based ADC}. (c) HIC chromatograms (214 nm) of ADCs based on mAb1 or mAb2, with designs C, A, and B (from top to bottom), containing LLAE (DBCO-C6-PEG8-VC-PABC-AE). The arrows indicate the corresponding DAR species in each sample. For the chromatograms presented here, 125 ng of mAb1 wt or mAb2 wt were added to the sample (5 μg) as internal standard before injection.

Figure 5

In vitro efficacy. Cytotoxic activity of (a) SLAE-based ADCs, (b) LLAE-based ADCs, (c) mAb wt, drug-linkers, and isotype ADC on high antigen-expressing cancer cell lines. Cytotoxic activity of (d) SLAE-based ADCs, (e) LLAE-based ADCs, (f) mAb wt and drug-linkers on low antigen-expressing cancer cell lines. The means and the standard deviations are the results of 3 independent experiments performed in triplicates.

Figure 6

Relative hydrophobicity . (a) Formula of the relative hydrophobicity (RH). For the determination of the relative hydrophobicity, 125 ng of mAb wt were added to the sample (2 μg) as internal standard before injection into the HIC-UV system. Relative hydrophobicity was calculated with design A-based ADC sharing the same antibody and the same drug or fluorophore as reference. A low RH value indicates a low hydrophobicity. (b) Schematic representations of the antibody sequence with AECK introduced at position HC-118 (mAbDesignA), the antibody sequence with AzF introduced at position HC-118 (mAbDesignD), the antibody sequence with AECK introduced at position HC-119 (mAbDesignE), and the chemical structure of Cy5 (DBCO-Cyanine5). Amino acids are numbered according to IgG1-Eu described by Edelman et al. [19]. (c) Relative hydrophobicity (RH) of the generated ADCs. RH of design A-based ADCs, which are equal to 1 by definition, are shown in Table S1.