Fiona Chionh

The Eph receptor tyrosine kinase EphA3 is over-expressed in a range of tumours, most commonly in the tumour microenvironment (TME), including in immunosuppressive cancer-associated fibroblasts (CAFs). In contrast, normal homeostatic tissues show little or no expression of EphA3, making it an attractive therapeutic target. We previously developed a humanized EphA3 antibody (Ifabotuzumab) which was tested in preclinical models and in phase I clinical trials, showing tumour specificity and biological activity. To improve its efficacy, we have tested novel antibody-drug conjugates (ADCs) of Ifabotuzumab with the microtubule disruptor monomethyl auristatin E (MMAE), the Topoisomerase I inhibitor deruxtecan (DXd), and the DNA binding cytotoxin pyrrolobenzodiazepine dimer (PBD), using cleavable linkers and site-specific ‘AJICAP’ peptide-mediated conjugation. In vitro analysis showed retention of specific binding of Ifabotuzumab-ADCs to EphA3 and specific, dose-dependent killing of EphA3-expressing cells. Ifabotuzumab-PBD showed the highest in-vitro potency and was alone effective against EphA3-expressing cancer-associated fibroblasts. Treatment of mice bearing EphA3-expressing tumour xenografts with all Ifabotuzumab-ADCs decreased tumour growth and increased survival, again with Ifabotuzumab-PBD the most efficacious. Ifabotuzumab-PBD was also able to inhibit growth of tumours that express EphA3 only in stromal cells in the TME, using syngeneic tumour models in immunocompetent mice. Notably, combination with the immune-checkpoint inhibitor anti-PD1 increased CD8+ T cells in tumours, and improved tumour inhibition, compared to anti-PD1 alone. This data indicates that Ifabotuzumab-ADCs have potential as novel specific anti-tumour agents capable of targeting the TME in solid tumours.

Purpose:Anti–PD-1/PD-L1 blockade combined with chemotherapy has become the first-line treatment for advanced biliary tract cancers. Combined anti–PD-1/CTLA-4 blockade using nivolumab and ipilimumab has shown encouraging activity in patients with intrahepatic cholangiocarcinoma (iCCA) and gallbladder carcinoma (GBC) in two trials (CA209–538 and SWOG1609). MoST-CIRCUIT further evaluated combined checkpoint blockade using nivolumab/ipilimumab in patients with advanced iCCA and GBC. Patients and Methods:Patients with a maximum of 1 line of prior systemic therapy were enrolled as cohort B into MoST-CIRCUIT, a single-arm, nonrandomized phase 2 trial. Patients received nivolumab 3 mg/kg and ipilimumab 1 mg/kg every 3 weeks for four doses, followed by nivolumab 480 mg every 4 weeks for 96 weeks. Response (RECIST 1.1) was assessed every 12 weeks. Coprimary endpoints were objective response rate (ORR) and 6-month progression-free survival (6-PFS), with the secondary endpoints being median overall survival (mOS), PFS, and treatment-related toxicity. Results:Sixty patients (37 iCCA and 23 GBC) were enrolled; 85% were pretreated, including 13 patients with durvalumab. The ORR was 12% (2% complete response, 10% partial response): 3% and 26% in the iCCA and GBC subgroups, respectively. The 6-PFS was 27% (iCCA 19%; GBC 39%), and mOS was 7 months. In the immunotherapy-naïve population, the ORR was 19% (iCCA 10%; GBC 38%). Severe immune-related adverse events were observed in 20% of patients. Conclusions:Efficacy was limited in what is the largest biliary tract cancer cohort treated to date with combined anti–CTLA-4/PD-1 blockade. Encouraging activity was observed in the GBC subgroup. Further evaluation of checkpoint inhibition in biliary tract cancer should focus on patients with GBC.

Representativeness of the Study population.

Purpose: Anti–PD-1/PD-L1 blockade combined with chemotherapy has become the first-line treatment for advanced biliary tract cancers. Combined anti–PD-1/CTLA-4 blockade using nivolumab and ipilimumab has shown encouraging activity in patients with intrahepatic cholangiocarcinoma (iCCA) and gallbladder carcinoma (GBC) in two trials (CA209–538 and SWOG1609). MoST-CIRCUIT further evaluated combined checkpoint blockade using nivolumab/ipilimumab in patients with advanced iCCA and GBC. Patients and Methods: Patients with a maximum of 1 line of prior systemic therapy were enrolled as cohort B into MoST-CIRCUIT, a single-arm, nonrandomized phase 2 trial. Patients received nivolumab 3 mg/kg and ipilimumab 1 mg/kg every 3 weeks for four doses, followed by nivolumab 480 mg every 4 weeks for 96 weeks. Response (RECIST 1.1) was assessed every 12 weeks. Coprimary endpoints were objective response rate (ORR) and 6-month progression-free survival (6-PFS), with the secondary endpoints being median overall survival (mOS), PFS, and treatment-related toxicity. Results: Sixty patients (37 iCCA and 23 GBC) were enrolled; 85% were pretreated, including 13 patients with durvalumab. The ORR was 12% (2% complete response, 10% partial response): 3% and 26% in the iCCA and GBC subgroups, respectively. The 6-PFS was 27% (iCCA 19%; GBC 39%), and mOS was 7 months. In the immunotherapy-naïve population, the ORR was 19% (iCCA 10%; GBC 38%). Severe immune-related adverse events were observed in 20% of patients. Conclusions: Efficacy was limited in what is the largest biliary tract cancer cohort treated to date with combined anti–CTLA-4/PD-1 blockade. Encouraging activity was observed in the GBC subgroup. Further evaluation of checkpoint inhibition in biliary tract cancer should focus on patients with GBC.

Supplementary Figure 1 shows the synergistic effect of trametinib plus panobinostat in CRC cell lines and PDTO's

Supplementary figure 4 shows phosphorylated ERK staining in an adenoma from a ACDX2 mouse

Supplementary figure 2 shows the effect of trametinib plus panobinostat on colony formation in HCT 116 cells

Supplementary figure 5 shows mouse weight during treatment in two of the mouse studies

Supplementary figure 3 shows combining trametinib with different classes of HDAC inhibitors in COLO 201 cells