Background Clustering of CD27 on the plasma membrane of T cells induces T-cell activation, proliferation, and differentiation. Therefore, this costimulatory receptor represents a target for cancer immunotherapy. Multiple monoclonal antibodies (mAbs) targeting CD27 are being explored in the clinic, which require Fc gamma receptor (FcγR)-mediated crosslinking to induce CD27 agonism. HexaBody-CD27 (GEN1053/BNT313) is a novel anti-CD27 mAb with an IgG Fc domain engineered to induce CD27 agonist activity independently of FcγR-bearing cells, which may be scarce in tumors. The Fc domain was further modified to silence Fc-mediated antibody effector functions, with the aim to prevent T-cell depletion. Here we present preclinical characterization of the mechanism of action of HexaBody-CD27.

Methods Target binding characteristics and functional activity of HexaBody-CD27 were analyzed in vitro using flow cytometry, cell-based reporter assays and primary human lymphocyte assays. The capacity of HexaBody-CD27 to induce tumor-infiltrating lymphocyte (TIL) proliferation was assessed ex vivo using non-small cell lung cancer (NSCLC) tissue resected from patients. HexaBody-CD27 activity in vivo was investigated in human CD27 knock-in mice that were immunized with ovalbumin and treated with HexaBody-CD27 by characterizing peripheral blood and splenic T cells using flow cytometry.

Results HexaBody-CD27 exhibited dose-dependent CD27 agonist activity in reporter assays, independent of crosslinking via FcγR-expressing cells. In contrast, agonist activity of benchmark anti-CD27 antibody analogs was dependent on FcγR-mediated crosslinking. HexaBody-CD27 did not functionally engage with FcγRs, and membrane-bound HexaBody-CD27 was unable to bind C1q, confirming functional silencing of the IgG Fc domain. In vitro, HexaBody-CD27 enhanced activation, proliferation, and proinflammatory cytokine secretion of TCR-stimulated human CD4⁺ and CD8⁺ T cells as well as CD8⁺ T-cell mediated cytotoxic activity towards cognate antigen-expressing tumor cells. In TIL assays with human NSCLC tumor tissue, HexaBody-CD27 promoted expansion of CD8⁺ T cells. In human CD27 knock-in mice, HexaBody-CD27 enhanced expansion and IFN-γ secretion of antigen-specific CD8⁺ T cells. No decrease in percentages of circulating or splenic T cells was detected in vivo after treatment with HexaBody-CD27, whereas treatment with a benchmark anti-CD27 mAb analog resulted in a marked reduction of T cells.

Conclusions HexaBody-CD27 has a functionally inert Fc domain and exhibits FcγR-crosslinking-independent CD27 agonist activity, a unique mechanism of action that distinguishes HexaBody-CD27 from benchmark mAbs targeting CD27. In preclinical studies in vitro and in vivo, HexaBody-CD27 increased T-cell activation, proliferation, cytokine secretion, and cytotoxic activity. A first-in-human clinical trial has been initiated to evaluate HexaBody-CD27 in patients with advanced solid tumors (NCT05435339).

Ethics Approval The use of resected tumor tissue was approved by BioNTech SE‘s Ethics Board, approval number 837.309.12 (8410-F).

All mouse studies were performed at Crown Bioscience Inc. in China. Animals were housed and handled in accordance with good animal practice as defined by the regulations of the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC).