A plug-and-play approach to antibody-based therapeutics via a chemoselective dual click strategy

Abstract

Although recent methods for the engineering of antibody-drug conjugates (ADCs) have gone some way to addressing the challenging issues of ADC construction, significant hurdles still remain. There is clear demand for the construction of novel ADC platforms that offer greater stability, homogeneity and flexibility. Here we describe a significant step towards a platform for next-generation antibody-based therapeutics by providing constructs that combine site-specific modification, exceptional versatility and high stability, with retention of antibody binding and structure post-modification. The relevance of the work in a biological context is also demonstrated in a cytotoxicity assay and a cell internalization study with HER2-positive and -negative breast cancer cell lines.

Figure 1. Approaches used for the generation of FDA-approved ADCs.

(a) Lysine modification leading to a heterogeneous product mixture, undefined physical and pharmacokinetic properties, and wide distribution of drug-to-antibody ratio. (b) Modification of reduced disulfide bonds leading to a heterogeneous product mixture, loss of structural disulfide bonds and a suboptimal therapeutic index.

Figure 2. Functional disulfide re-bridging followed by a dual click approach.

Disulfide re-bridging by a pyridazinedione construct yields a site-selectively modified antibody with dual modalities.

Figure 3. Properties of dibromopyridazinediones and structure of Astra-PD 1.

(a) General properties of the pyridazinedione platform. (b) Chemical structure of Astra-PD 1 with an alkyne and a strained alkyne handle.

Figure 4. Sequential modifications from native Fab-Her 2 to afford Fab-Astra-PEG4-Cy5 5.

Re-bridging of Fab-Her 2 and regioselective dual modification using SPAAC with PEG₄-N₃ followed by CuAAC with Sulfo-Cy5-N₃ to yield Fab-Astra-PEG₄-Cy5 5 conjugate as a single product.

Figure 5. Activity and stability data for model construct Fab-Diet 6.

(a) Structure of Fab-Diet 6. (b) Binding activity data for native Fab-Her 2 and Fab-Diet 6 assessed by ELISA. (c) SDS–PAGE gel following incubation of Fab-Diet 6 in blood plasma mimicking conditions for 0, 1, 2, 3, 5 and 7 days (lanes 1–6, respectively). (d) SDS–PAGE gel of Fab-Diet 6, Fab-Diet 6 after 8 months of storage at 4 °C in PBS, Fab-Diet 6 after 24 h at 37 °C in buffer pH 3.1 and Fab-Diet 6 after 24 h at 37 °C in buffer pH 9.0 (lanes 1–4, respectively). (e) Deconvoluted MS data of Fab-Diet 6 after 8 months of storage at 4 °C in PBS. (f) Deconvoluted MS data of Fab-Diet 6 after 24 h at 37 °C in buffer pH 3.1. (g) Deconvoluted MS data of Fab-Diet 6 after 24 h at 37 °C in buffer pH 9.0.

Figure 6. Construction of Fab-Astra-Dox-PEG_20k 7, a fragment-based ADC with an additional lifetime extension modality.

(a) SDS–PAGE analysis for bioconjugates Fab-Astra-Dox-PEG_20k 7 and Fab-Astra-Dox (lanes 1 and 2, respectively). (b) Structure of bioconjugate Fab-Astra-Dox-PEG_20k 7. (c) Deconvoluted MS data of Fab-Astra-Dox.

Figure 7. Construction of Her-Astra-Dox-Cy5 8, a dually modified ADC with both a cytotoxic drug and a fluorophore.

(a) Structure of fully re-bridged and dually functionalized Herceptin. (b) SDS–PAGE analysis of Her-Astra, Her-Astra-Dox, Her-Astra-Dox-Cy5 8 and Herceptin (lanes 1–4, respectively).

Figure 8. Binding activity of dually modified constructs by ELISA.

Binding activity of Fab-Her 2, Fab-Astra-Dox-PEG_20k 7, Her-Astra-Dox-Cy5 8 and unmodified full antibody Herceptin (Her).

Figure 9. Internalization study of fragment-based and full length antibody-based bioconjugates.

(a) BT-474 breast cancer cells were treated at 4 °C with Her-PD-AlexaFluor488 and Fab-PD-AlexaFluor488 (green); both constructs bound at this temperature and were found inside the cells once internalization was allowed by incubating at 37 °C for 1 h. (b) No signal from fluorophore-conjugated constructs Her-PD-AlexaFluor488 and Fab-PD-AlexaFluor488 was detected when allowed to bind to the MDA-MB-468 breast cancer cells.

Figure 10. Inhibition of cell proliferation in cancer cell lines with different levels of HER2 expression.

(a) BT-474 (HER2-positive) and MDA-MB-468 (HER2-negative): Dox alone; IC₅₀=39 and 10 nM for BT-474 and MDA-MB-468, respectively. (b) BT-474: Fab-Astra-Dox-PEG_20k 7, Her-Astra-Dox-Cy5 8 (at similar concentration in Dox) in comparison with Herceptin and Fab-Her 2; IC₅₀=2.8 and 2.1 μM for conjugates 7 and 8, respectively (c) MDA-MB-468: Fab-Astra-Dox-PEG_20k 7, Her-Astra-Dox-Cy5 8 (at similar concentration in Dox) in comparison with Herceptin and Fab-Her 2.