The Feasibility Study of Legumain-Cleavable Linkers for the Efficient and Selective Activation of ADCs with KSPi Payloads

The Importance of Developing Novel Cleavable Linkers

Despite the rapid development of ADCs, some ADCs have failed to achieve a large enough therapeutic window in patients due to dose-limiting toxicity caused by nonspecific payload release in circulation. This is an urgent need for developing novel cleavable linkers with improved tumor specificity to ensure high activity release of payloads in cancer tissues and low ADC uptake into healthy organs.

Creative Biolabs provides one-stop ADC development services with complete and scientific solutions according to your requirements.

In this study, scientists used trastuzumab, a HER2-targeting monoclonal antibody (mAb), and ITEM-4, an antagonist antibody against TWEAKR, as antibodies to design and synthesize legumain-cleavable KSPi anti-TWEAKR ADCs 6a−6h and anti-HER2 ADCs 7a-7h by utilizing a novel linker that is efficiently and selectively cleaved by the tumor-associated protease legumain.

Synthesis of Legumain-Cleavable KSPi ADCs 7a−h and ADCs 6a−h

The partially protected KSPi intermediate 1 attachment of an orthogonally protected dipeptide, removal of the Teoc protecting group, attachment of legumain-cleavable peptides, and final removal of all protecting groups provided the intermediates 3a−h. These molecules were reacted with 1,1′-[(1,5-dioxopentane-1,5-diyl)bis(oxy)] dipyrrolidine-2,5-dione to yield the activated intermediates 4a−h. Subsequently, activated intermediates 4a−h, respectively conjugated with trastuzumab and ITEM-4 via its lysine side chains to synthesize ADCs 7a−h and ADCs 6a−h.

Furthermore, small molecules 5a, 5b, 5c, and 5e corresponding to the KSPi-linker derivatives of respective ADCs (6a, 6b, 6c, 6e, and 7a, 7b, 7c, 7e) were also synthesized (Fig.1).

Fig. 1. The Synthesis of Small Molecule Tool Compounds 5a, 5b, 5c, and 5e and Legumain-Cleavable AntiTWEAKR ADCs 6a−6h and Anti-HER2 ADCs 7a-7h. (Lerchen HG, et al., 2020)

At Creative Biolabs, we have successfully developed an advanced "DrugLnk" services platform for drug-linked complexes for ADC development projects and provide one-stop antibody conjugation services.

Feasibility Analysis

In this study, the feasibility of novel cleavable linkers was evaluated and verified through in vitro cytotoxicity, payload release test, and the therapeutic potential of the legumain-cleavable KSPI ADC in vivo.

In Vitro Cytotoxicity

• The potency against the target-expressing cell lines of ADC 6a-h and ADC 7a-h was studied, respectively. Both the anti-TWEAKR ADC 6a and the anti-HER2 ADC 7a with legumain substrate sequence alanine−alanine−asparagine (Ala-Ala-Asn) demonstrated high potency against the target-expressing cell lines, while the respective isotype control ADCs showed no activity (>300 nM), demonstrating the essential role of L-configured Asn at the legumain cleavage site for the release of the active metabolite.
• Similarly, ADCs 6b, 6c, 7b, and 7c, which substituted the central alanine residue with unnatural Dalanine or N-methyl alanine, showed similar potency to ADCs 6a and 7a in all tested cell lines.
• Based on these findings, the Asn residue essential for legumain cleavage was further replaced with close analogs such as D-Asn (6f, 7f), isosteric Leu (6g, 7g), and homologous Gln (6h, 7h). The ADCs 6e and 7e were found to be equipotent with the respective ADCs with tripeptide linkers, indicating that the cleavage indeed does not occur in the absence of the Asn residue at the cleavage site.

Payload Release Test

• The researchers conducted investigations on the release of the active metabolite 8 from different biochemical assays using small molecule payload-linker prodrugs 5a-5e. In the biochemical assay for legumain-mediated cleavage, it was observed that 5a-5e produced a significant amount of the active metabolite 8, indicating a high specificity for legumain-mediated cleavage.
• Based on this finding, NCI-H292 tumor cells were exposed to similar anti-TWEAKR ADCs 6b, 6c, and 6e that contained the agonistic anti-TWEAKR mAb TPP-2658. It was discovered that all three ADCs released similar levels of active metabolites 8 in the cellular fraction (Fig. 2).

Fig. 2. Concentrations of the active metabolite 8 released from the ADCs 6b, 6c, and 6e with an anti-TWEAKR mAb TPP-2658. (Lerchen HG, et al., 2020)

• The cleavage and metabolite formation were further evaluated in lysosomal preparations obtained from rat hepatocytes, and found that the positive control (cathepsin-cleavable ADC) showed ~85% release of active metabolite 8. In contrast, the legumain-cleavable ADCs 6a-c and 6e are relatively stable.

Fig. 3. Metabolite formation from the ADCs 6a−c, e, and a cathepsin B-cleavable control ADC after incubation with a lysosomal extract from rat liver for 48 h. (Lerchen HG, et al., 2020)

In Vivo Efficacy Assessment

The researchers compared the ADCs 6b, 6c, and 6e with different substrate sequences side-by-side in mouse models of NCIH292 NSCLC and Ku-19-19 urothelial cancer. The results (Fig. 4) demonstrated that administering twice weekly doses of 5 mg/kg of ADCs 6b, 6c, and 6e can lead to sustained regression of NCI-H292 tumors. Similarly, three weekly doses of the ADCs at 5 mg/kg also exhibited high efficacy and selectivity in the Ku19-19 model.

Fig. 4. Efficacy of the anti-TWEAKR ADCs 6b, 6c, and 6e with an antagonistic TWEAKR mAb ITEM4 and the respective isotype control ADCs in NCI-H292 and Ku-19-19 tumor-bearing NMRI nude mice. (Lerchen HG, et al., 2020)

Reference

1. Lerchen HG, Stelte-Ludwig B, Sommer A, et al. Tailored Linker Chemistries for the Efficient and Selective Activation of ADCs with KSPi Payloads. Bioconjug Chem. 2020, 31(8):1893-1898.

For Research Use Only. NOT FOR CLINICAL USE.