BT8009; A Nectin-4 Targeting Bicycle Toxin Conjugate for Treatment of Solid Tumors

Abstract

Multiple tumor types overexpress Nectin-4 and the antibody-drug conjugate (ADC), enfortumab vedotin (EV) shows striking efficacy in clinical trials for metastatic urothelial cancer, which expresses high levels of Nectin-4, validating Nectin-4 as a clinical target for toxin delivery in this indication. Despite excellent data in urothelial cancer, little efficacy data are reported for EV in other Nectin-4 expressing tumors and EV therapy can produce significant toxicities in many patients, frequently leading to discontinuation of treatment. Thus, additional approaches to this target with the potential to extend utility and reduce toxicity are warranted. We describe the preclinical development of BT8009, a "Bicycle Toxin Conjugate" (BTC) consisting of a Nectin-4-binding bicyclic peptide, a cleavable linker system and the cell penetrant toxin mono-methylauristatin E (MMAE). BT8009 shows significant antitumor activity in preclinical tumor models, across a variety of cancer indications and is well tolerated in preclinical safety studies. In several models, it shows superior or equivalent antitumor activity to an EV analog. As a small hydrophilic peptide-based drug BT8009 rapidly diffuses from the systemic circulation, through tissues to penetrate the tumor and target tumor cells. It is renally eliminated from the circulation, with a half-life of 1-2 hours in rat and non-human primate. These physical and PK characteristics differentiate BT8009 from ADCs and may provide benefit in terms of tumor penetration and reduced systemic exposure. BT8009 is currently in a Phase 1/2 multicenter clinical trial across the US, Canada, and Europe, enrolling patients with advanced solid tumors associated with Nectin-4 expression.

Graphical abstract

Figure 1.

High content imaging showing anti-MMAE mAb staining after incubation of MDA-MB-468 cells with test agent (each at 1 μmol/L) n = 4–8; A, Composite showing nuclei stained with DAPI (Cyan), membrane marker (purple) anti-MMAE mAb (green); B, with 1 μmol/L Nectin-4 ADC; C, with 1 μmol/L BT8009; D, with 1 μmol/L MMAE; E, with non-binding BTC (BCY8781).

Figure 2.

PK profile of BT8009 and MMAE in three mouse xenograft models. MMAE is rapidly cleared from plasma but retained in tumor substantially longer. Error bars indicate SD of n = 3 per timepoint.

Figure 3.

BT8009 shows dose-related antitumor activity in; A, A CDX (triple-negative breast cancer) xenograft model. Dosing of the 3 mg/kg initial treatment group was ceased on day 42. Vehicle-treated animals were switched to 3 mg/kg BT8009 qw on day 40 and then 5 mg/kg qw on day 75. B, a PDX (non–small cell lung) xenograft model. Dosing of the 3 mg/kg initial treatment group was ceased on day 45. Vehicle-treated animals were switched to 3 mg/kg BT8009 qw on day 36. Tumor volumes are shown as mean ± standard error of the mean (n = 3–5) and statistical analysis performed with ordinary one-way ANOVA with Tukey's post hoc test for multiple comparisons ***, P < 0.001 and ****, P < 0.0001.

Figure 4.

Relationship between BT8009 antitumor activity and target expression in CDX and PDX models; A, The percentage of change from initial tumor volume (blue columns–values above 100% indicate tumor growth, below 100% indicate tumor regression) and Nectin-4 expression assessed by FACS (orange circles, indicating antigen-binding sites per cell). FACS and IHC data are not available for 4 PDX models on right of A. IHC for Nectin-4 (stained brown) is shown in panels below the cell type; B, The percentage of change from initial tumor volume and Nectin-4 expression, assessed by IHC, in 14 NSCLC PDX models. Animals were dosed with 3 mg/kg BT8009, qw, in all studies.

Figure 5.

A, In the MDA-MB-468 xenograft BT8009 was dosed alone or in combination with an excess of unconjugated bicycle, BCY8234, antitumor activity was attenuated by the unconjugated peptide. BT8009 showed significant difference from vehicle from day 7 onwards, BT8009 with BCY8234 showed significant difference from vehicle from 14 days onwards. B, In the same study the non-binding BTC homologue BCY8781 failed to produce comparable effect with BT8009, with significant difference from vehicle from day 9 onwards. BT8009 versus BCY8781 were significantly different on days 18 and 21. C–E, In the LU-01–0412 PDX model, BT8009 consistently showed greater tumor regression than the non-binding homologue BCY8781 at equivalent doses. Tumor volumes are shown as mean ± standard error of the mean (n = 3–5) and statistical analysis performed with ordinary one-way ANOVA with Tukey's post hoc test for multiple comparisons n.s., non-significant; * P < 0.05; ***P < 0.001; and **** P < 0.0001.

Figure 6.

Head-to-head studies with the Nectin-4 ADC. A and B, Dose response to ADC in NSCLC CDX (NCI-H292) with high dose showing slower tumor regression than with BT8009; C, Response in TNBC CDX (MDA-MB-468) showing slower response to treatment with ADC; D, Equivalent regression rate with ADC, but tumor growth resumed on cessation of dosing, indicating incomplete regression in a lung PDX (LU-01–0412), unlike after BT8009; E, Lack of effect of ADC in head and neck PDX (HN-13–001) compared with BT8009 efficacy. ADC was dosed at 5 mg/kg on D0 and D7, then increased to 10 mg/kg on D14. ADC-treated tumors remain responsive to BT8009. Tumor volumes are shown as mean ± standard error of the mean (n = 3–16) and statistical analysis performed with ordinary one-way ANOVA with Tukey's post hoc test for multiple comparisons n.s., non-significant; *, P < 0.05; ***, P < 0.001; and ****, P < 0.0001.