SC134-deruxtecan, a fucosyl-GM1 targeting ADC for small cell lung cancer therapy

Abstract

Background: Small cell lung cancer (SCLC) remains a difficult disease to treat with poor long-term survival rates. New therapies offer modest overall survival benefit beyond that of chemotherapy alone, necessitating the development of improved therapies. Fucosyl-GM1 (FucGM1) is a glycolipid highly expressed on SCLC cells, but virtually absent in normal tissues, suggesting strong potential for targeted therapy. We have developed SC134-deruxtecan, an antibody drug conjugate (ADC) targeting FucGM1 in SCLC, and characterized its preclinical activity.

Methods: SC134 binding specificity and affinity were tested through enzyme-linked immunosorbent assay (ELISA), surface plasmon resonance (SPR), flow cytometry against several SCLC cell lines, and immunohistochemistry (IHC) of clinical samples and animal tissues. In silico modelling supplemented the FucGM1 binding specificity. Internalization kinetics and colocalization of SC134 with the lysosomes were investigated through imaging flow cytometry. Direct cytotoxicity as well as bystander killing and antibody dependent cell cytotoxicity (ADCC) by SC134-deruxtecan were determined using in vitro cell cytotoxicity assays. SC134-deruxtecan's efficacy was evaluated in vivo using a DMS79 xenograft model.

Results: SC134 specifically targets FucGM1, without GM1 cross-reactivity, with nanomolar affinity. In silico modelling of the SC134 FucGM1 binding site revealed a relatively narrow binding pocket, occupied by the terminal three glycans with multiple Fucose-engaging interactions. Robust FucGM1 expression in frozen SCLC patient tissues was evident, whilst tissue cross-reactivity analysis indicated non-human primates as well as mice as suitable tox models. FucGM1 binding by SC134 led to effective internalization, with a 6.9-h half-life, lysosomal colocalization, culminating in sub-nanomolar drug delivery efficiency, across a range of payloads. Covalent deruxtecan conjugation of SC134 with a DAR 8 and a cleavable linker showed effective (nanomolar) in vitro killing of SCLC cell lines such as DMS79 and DMS153, with concentration-dependent bystander killing of FucGM1-negative AGS cells. SCLC cell killing was further augmented through ADCC. Potent in vivo DMS79 xenograft killing was seen at 3mg/kg SC134-deruxtecan, which was well tolerated.

Conclusion: The tumour-specific nature of FucGM1, combined with the potent SCLC killing by SC134-deruxtecan underscore the development potential of SC134 for use as an ADC therapy against SCLC.

Keywords: ADC; Fucosyl-GM1; Glycolipid; Internalization; Small cell lung cancer.

Fig. 1

SC134 exhibits avid FucGM1 binding, facilitated by a narrow binding pocket A FucGM1 cartoon with sphingosine and hydrophobic lipid tail, the importance of the Fucose residue in binding is highlighted; B ELISA binding of FucGM1 (ceramide and BSA conjugate) to SC134, absence of GM1 cross reactivity; C Biacore analysis of real-time SC134 binding to FucGM1-BSA compared to GM1a-BSA); D In silico modelling of FucGM1 glycolipid binding to SC134

Fig. 2

FucGM1 exhibits a superb differential tumour versus normal tissue distribution and is expressed in preclinical toxicology models A Comparative H-scores for DLL3 expression by PDX tissues (Supplemental Fig. 3A) compared to FucGM1 from [22]; B Comparative H-scores and staining for FucGM1, DLL3 and TTF-1 in patient SCLC samples (patient details in Supplemental Table 1); C H-score distribution of FucGM1 in squamous NSCLC tissues (staining in Supplemental Fig. 3C); D FucGM1 positive mouse tissues: (A) breast, (B) colon, (C) skeletal muscle, (D) pancreas, (E) skin, (F) small intestine (G) stomach (H) testes, (I) thymus, (J) Pituitary (C57BL/6); E FucGM1 positive rat tissues (A) mesothelium, (B) stomach (foveolar cells), (C) stomach (crypt cells); F FucGM1 positive cynomolgous monkey tissues: (A) thymus, (B) skin and (C) small intestine

Fig. 3

SC134 targets SCLC cell lines with nanomolar efficiency and displays efficient internalization A Flow cytometry analysis of SC134 cell binding to SCLC cell lines. Histograms show binding of 50 nM SC134 (DSM79, DMS53, H526) or 500 nM SC134 (DMS153) (black) against control (gray); B SC134 internalization on DMS79, anti-transferrin receptor antibody was included as a positive control; i. representative cellular fluorescence of AF647-SC134 or AF647-anti-transferrin receptor over time, out of approximately 500–1500 gated events. Image number corresponds to the recorded event acquisition number. Brightness is increased for transferrin receptor labelling compared to SC134 to help visualization of antibody location. This does not affect internalization values. ii. internalization score of each antibody over time. iii. Percentage of AF647-positive cells with a positive internalization score, over time. C Co-localization of SC134-AF647 (red) and lysosomal LAMP1 (green) following incubation of DMS79 cells in the presence of SC134-AF647 for 0 h (i) and 24 h (ii)

Fig. 4

SC134-deruxtecan shows potent in vitro SCLC cell killing, with bystander and effector cell mediated impact A Cell survival assay, following 72 h culture with SC134 and non-targeting rituximab control ADC using DMS79, DMS153, DMS53 and H526. B Bystander assay showing AGS cell number following 6-day co-culture with increasing DMS79 ratios and ADC concentrations for 6 days. AGS cell number was determined through flow cytometry and negative gating with SC134-AF647. C ADCC activity measured as percentage cytotoxicity following co-culture of DMS79 target cells with PBMC in the presence of SC134-deruxtecan or rituximab-deruxtecan control. DMS79 alone with SC134-deruxtecan was used as a negative control for ADCC

Fig. 5

Excellent in vivo tumour control against DMS79 xenograft by SC134-deruxtecan A Anti-tumour efficacy of SC134-deruxtecan against DMS79 xenograft. Mice (n = 10/group) were randomised on day 10 when tumour size was ~ 102 mm³. SC134-deruxtecan and rituximab-deruxtecan were dosed at 3 mg/kg on day 11 and 25. Symbols represent mean tumour volume + SEM. Mixed effects model with Geisser-Greenhouse correction used for statistical tests. ****p ≤ 0.0001. B Relative mean bodyweight over time, as a percentage of bodyweight at start of dosing (day 11), following dosage of SC134-deruxtecan and non-targeting Rituximab-deruxtecan control. Symbols represent mean bodyweight + SEM