Development of a facile antibody-drug conjugate platform for increased stability and homogeneity

Abstract

Despite the advances in the design of antibody-drug conjugates (ADCs), the search is still ongoing for novel approaches that lead to increased stability and homogeneity of the ADCs. We report, for the first time, an ADC platform technology using a platinum(ii)-based linker that can re-bridge the inter-chain cysteines in the antibody, post-reduction. The strong platinum-sulfur interaction improves the stability of the ADC when compared with a standard maleimide-linked ADC thereby reducing the linker-drug exchange with albumin significantly. Moreover, due to the precise conserved locations of cysteines, both homogeneity and site-specificity are simultaneously achieved. Additionally, we demonstrate that our ADCs exhibit increased anticancer efficacy in vitro and in vivo. The Pt-based ADCs can emerge as a simple and exciting proposition to address the limitations of the current ADC linker technologies.

Scheme 1. Comparison of conventional linker technologies with a Pt(ii)-linker. (a) Conjugation to lysines results in a heterogeneous mixture. (b) Conjugation to cysteines via maleimide leads to reduced higher order structural stability and instability in the presence of albumin. (c) Pt(ii)-linker re-bridges the antibody chains with strong Pt–S interaction imparting stability, homogeneity and site-specificity.

Scheme 2. Structure, design and synthesis of Pt–PEG–CPT. (Reagents and all intermediates are shown in the ESI; PEG: polyethyleneglycol.)

Fig. 1. SE-HPLC profiles of conjugates. Chromatogram of Ctx–Pt–PEG–CPT (a) at 280 nm showing low presence of aggregates (<1%) and (b) fluorescence of CPT corresponding to the antibody peak with negligible presence of free camptothecin (<1%). Chromatogram of Tra–Pt–PEG–CPT (c) at 280 nm showing low presence of aggregates (2%) and (d) fluorescence of CPT corresponding to the antibody peak with negligible presence of free camptothecin (<1%).

Fig. 2. Antigen-binding ability. ELISA demonstrating that the affinity of the antibody is not compromised upon Pt(ii)-based conjugation. (a) Ctx and Ctx–Pt–PEG–CPT binding to EGFR. (b) Tra and Tra–Pt–PEG–CPT binding to HER2. (c) Rtx and Rtx–Pt–PEG–CPT binding to CD20. (d) Calculated K _d values compared with reported binding affinities.^– Error bars represent standard error of the mean.

Fig. 3. HIC profiles of Tra_Fab–drug conjugates. (a) A representative HIC profile at 280 nm of Tra_Fab–Pt–PEG–CPT (DAR: 1.9 by UV), demonstrating > 95% conjugation efficiency and homogeneity. The DAR was calculated to be 1.9 by HIC. (b) A representative HIC profile at 280 nm of the purified peak of Tra_Fab–Pt–PEG–CPT (27.8 min). The DAR was verified by UV spectroscopy as 2. (c) A representative HIC profile at 280 nm of Tra_Fab–mal–PEG–CPT (DAR: 0.65 by UV) demonstrating heterogeneity with the presence of both DAR1 and DAR2 species. The DAR was calculated to be 0.59 by HIC. (1) Unconjugated Fab; (2) Tra_Fab–Pt–PEG–CPT (DAR: 2); (3) Tra_Fab–Pt–PEG–CPT (DAR: 2); (4) Tra_Fab–mal–PEG–CPT (DAR: 1); (5) Tra_Fab–mal–PEG–CPT (DAR: 2).

Scheme 3. ADC stability in the presence of albumin. (a) Maleimide linkers undergo a retro-Michael reaction regenerating the maleimide which can react with the free thiol present on albumin. (b) Pt(ii)-linker is significantly less reactive towards the albumin thiol.

Fig. 4. Stability in the presence of albumin. An overlay of the SE-HPLC fluorescence chromatogram of the albumin fraction post-incubation with (a) Ctx–Pt–PEG–CPT, (b) Ctx–mal–PEG–CPT, (c) Tra–Pt–PEG–CPT and (d) Tra–mal–PEG–CPT at 0 h and 72 h demonstrates that at least 4-fold more linker–drug attaches to albumin covalently in the case of mal–PEG–CPT when compared with Pt–PEG–CPT. (*): albumin peak; the peaks eluting post 8 min are unconjugated free low molecular weight species.

Fig. 5. Higher-order structural stability. SDS-PAGE gel (Coomassie-stained) under non-reducing conditions, showing that the antibody light chain and heavy chain are held together by strong forces via the Pt(ii) linker. Fluorescence corresponding to CPT can be visualized on the gel under UV light. (a) Cetuximab, (b) trastuzumab, (c) rituximab, (d) trastuzumab Fab and (e) cetuximab Fab.

Fig. 6. Plasma stability. The stability of Ctx–Pt–PEG–CPT and Tra–Pt–PEG–CPT in (a) human plasma and (b) mouse plasma demonstrates the superior stability of the Pt-based conjugation with extended half-lives (c).

Fig. 7. In vitro potency of ADCs. Cell viability was assessed using an MTT assay and IC₅₀ values were calculated. (a) The Ctx–Pt–PEG–CPT conjugate showed a 118-fold increase in anti-proliferative activity on EGFR-high cells (MDA-MB-468) as compared to EGFR-low cells (MCF7). (b) The Tra–Pt–PEG–CPT conjugate showed a 327-fold increase in anti-proliferative activity on HER2-high cells (SK-BR-3) as compared to on HER2-low (MDA-MB-453) cells. Error bars represent standard error of the mean for at least 3 independent experiments. (c) IC₅₀ values (drug-equivalent concentration of ADC) obtained for various cell lines demonstrate the specificity and potency of the ADCs. Standard errors reported from at least 3 independent experiments. *Extrapolated values.

Fig. 8. In vivo efficacy. Antitumour activity of Ctx–Pt–PEG–CPT was evaluated in an A549 xenograft model in SCID Beige mice. The mice were implanted with 5 × 10⁶ A549 cells and grouped as untreated (control), cetuximab (Ctx) or Ctx–Pt–PEG–CPT (ADC) on a dosing schedule of q4dx5 for 2 cycles at 20 mg kg^–1 of test article. (a) The Ctx–Pt–PEG–CPT treated animals showed significant differences in tumour volume compared with the control group (P < 0.016), with a tumour growth inhibition (TGI) of 55%. (b) No significant difference in body weight was observed across the groups. (c) Tumour sections examined using a TUNEL assay (TUNEL, TMR-Red; Hoechst, blue) show a higher number of apoptotic cells in ADC treated tumours. Scale bar, 20 μm. (d) The percentage of TUNEL-positive cells in ADC treated tumour sections is significantly higher than those observed for cetuximab treated samples. **p < 0.0001; *p < 0.005.