Background
Cancer associated fibroblasts (CAFs) are a critical pro-tumorigenic and immunosuppressive cell subset within the tumor microenvironment (TME) that promotes resistance to immune checkpoint inhibitors. Enrichment of fibroblast activation protein (FAP)-expressing CAFs correlates with poor immunotherapy response across multiple cancer types, identifying these cells as potential drivers of resistance. TGFβ, produced by both tumor and stromal cells in the TME, is a key factor in differentiating fibroblasts into FAP+ CAFs. However, no TGFβ inhibiting agents that selectively target CAFs have been clinically tested. Here, we present AGEN1721, a novel, Fc-enhanced bifunctional antibody that selectively targets FAP and neutralizes TGFβ via an optimized TGFβR2 TRAP moiety fused to an engineered Fc region to maximize effector functions.
Methods
The binding affinity of AGEN1721 to FAP-expressing cells was evaluated using in vitro assays. The ability of AGEN1721 to mediate antibody-dependent cellular cytotoxicity (ADCC) against FAP+ cells was assessed using primary natural killer (NK) cells as effectors. Reporter assays were used to evaluate the capacity of AGEN1721 to neutralize TGFβ-mediated SMAD signaling, epithelial-to-mesenchymal transition (EMT), and tumor cell invasion. The anti-tumor efficacy of an AGEN1721 mouse surrogate (AGEN1721ms) was evaluated in an orthotopic 4T1 mouse breast carcinoma and MC38 subcutaneous colon cancer models, both as a single agent and in combination with anti-PD-1 and/or anti-CTLA-4 antibodies. Pharmacodynamic biomarkers were assessed to validate the mechanism of action of AGEN1721.
Results
In primary human in vitro assays, AGEN1721 potentiated NK cell-mediated ADCC to deplete FAP+ cells independent of FcRIIIA genotype. Additionally, AGEN1721 potently neutralized TGFβ isoforms, thereby inhibiting TGFβ-mediated canonical SMAD signaling, EMT and tumor cell invasion. In orthotopic breast cancer 4T1 and colorectal cancer MC38 mouse models, AGEN1721ms combined with anti-PD-1 and/or anti-CTLA-4 to enhance primary and metastatic tumor control compared to single-agent treatments. Efficacy was associated with tumor stroma remodeling, including decreased frequency of FAP+ CAFs, collagen rearrangement, reduced α-smooth muscle actin (αSMA) expression, and increased T cell infiltration. Notably, AGEN1721ms increased the frequency of Ki67+ CD8 T cells, and redistributed T cells from the tumor periphery to the tumor core, converting the tumors from an immune-excluded to an immune-inflamed phenotype.
Conclusions
AGEN1721 is a first-in-class bifunctional antibody designed to overcome immune exclusion and suppression mediated by CAFs and TGFβ in the tumor microenvironment. By concurrently depleting FAP+ CAFs and inhibiting TGFβ, AGEN1721 is designed to remodel the tumor stroma, enhance T cell infiltration, and improve responses to cancer immunotherapy.
Ethics Approval
This study obtained ethics approval for animal studies. IUCAC protocol numbers NLS18-016 and NLS21-015.