Anti-Extra Domain B Splice Variant of Fibronectin Antibody-Drug Conjugate Eliminates Tumors with Enhanced Efficacy When Combined with Checkpoint Blockade

Abstract

Extra domain B splice variant of fibronectin (EDB+FN) is an extracellular matrix protein (ECM) deposited by tumor-associated fibroblasts, and is associated with tumor growth, angiogenesis, and invasion. We hypothesized that EDB+FN is a safe and abundant target for therapeutic intervention with an antibody-drug conjugate (ADC). We describe the generation, pharmacology, mechanism of action, and safety profile of an ADC specific for EDB+FN (EDB-ADC). EDB+FN is broadly expressed in the stroma of pancreatic, non-small cell lung (NSCLC), breast, ovarian, head and neck cancers, whereas restricted in normal tissues. In patient-derived xenograft (PDX), cell-line xenograft (CLX), and mouse syngeneic tumor models, EDB-ADC, conjugated to auristatin Aur0101 through site-specific technology, demonstrated potent antitumor growth inhibition. Increased phospho-histone H3, a pharmacodynamic biomarker of response, was observed in tumor cells distal to the target site of tumor ECM after EDB-ADC treatment. EDB-ADC potentiated infiltration of immune cells, including CD3+ T lymphocytes into the tumor, providing rationale for the combination of EDB-ADC with immune checkpoint therapy. EDB-ADC and anti-PD-L1 combination in a syngeneic breast tumor model led to enhanced antitumor activity with sustained tumor regressions. In nonclinical safety studies in nonhuman primates, EDB-ADC had a well-tolerated safety profile without signs of either on-target toxicity or the off-target effects typically observed with ADCs that are conjugated through conventional conjugation methods. These data highlight the potential for EDB-ADC to specifically target the tumor microenvironment, provide robust therapeutic benefits against multiple tumor types, and enhance activity antitumor in combination with checkpoint blockade.

Figure 1.

EDB+FN is expressed in the ECM of multiple tumor types with restricted expression in normal tissues. A, Panels of breast cancers, NSCLCs, ovarian cancers, head and neck cancers, and pancreatic cancers were IHC stained for EDB+FN, demonstrating widespread ECM and stromal reactivity in the majority of tumors (inset table). B–G, Normal human tissues including heart (B), kidney (C), liver (D), lung (E), small intestinal muscularis mucosae (F), and muscularis externa (G) were stained for EDB+FN. Micron bar = 300 μm.

Figure 2.

EDB-ADC eliminates tumors in vivo in multiple tumor models. A, EDB+FN protein expression by IHC in H1975 CLX, NSCLC PDX, and pancreatic PDX models showing widespread ECM and stromal reactivity mirroring expression in human tumors. B, EDB-ADC induces TGI and complete tumor regressions in H1975 CLX, NSCLC PDX, and pancreatic PDX models in vivo. Mice bearing indicated human tumors were treated q4dx4 with EDB-ADC or controls at indicated dose levels on Days 0, 4, 8, 12 after tumors reached an average staging volume.

Figure 3.

EDB-ADC localizes to the tumor microenvironment and induces mitotic arrest in tumor cells. Pharmacodynamic IHC to interrogate mechanism of action of EDB-ADC in H1975 tumors 48 hours after a single 3 mg/kg ADC dose, demonstrating ADC distribution (top row as visualized by human IgG antibody IHC) and auristatin payload distribution (middle row, anti-auristatin IHC) in the tumor stromal/ECM compartment, and downstream increase in phospho-histone H3 (pHH3) expression (bottom row). Micron bar = 300 μm.

Figure 4.

EDB-ADC eliminates tumors in immunocompetent syngeneic mice & results in increased T cell Infiltration and PD-L1 upregulation. A, EDB+FN expression by IHC in EMT6 tumors. B, TGI with rcEDB-ADC in EMT6 mice. Mice bearing EMT6 tumors were treated q4d with rcEDB-ADC or vehicle controls at indicated dose levels for 1, 2, or 3 doses as indicated. C, EMT6 tumors treated with EDB-ADC were harvested 48 hours after the second dose of 3 mg/kg for pharmacodynamic IHC. H&E staining shows tumor cell lysis and acellular regions (top row), whereas IHC demonstrated increased CD3 infiltration (middle row) and upregulation of PD-L1 (bottom row). Micron bar = 200 μm.

Figure 5.

EDB-ADC demonstrates a combination benefit with checkpoint blockade. A, Combination of rcEDB-ADC at 3 mg/kg with anti-PD-L1 10 mg/kg showed a trend towards a combination benefit with 9 of 10 tumors completely regressing as compared with single agents in EMT6 tumor bearing mice. CR = complete regressions. B, Kaplan–Meier curves for a time to event analyses in EMT6 tumor bearing mice, showed effectiveness of combining rcEDC-ADC with anti-PD-L1 compared with single agents on % of tumors <3-fold from their first measurement as an indicator of the impact of the combination on tumor growth. C, Mice that had complete regressions in the initial study were rechallenged with subcutaneous injection of EMT6 cells and monitored for tumor growth. Mice previously treated with rcEDB-AC plus anti-PD-L1 were resistant to regrowth of new tumors that was significantly different from rcEDB-ADC alone.