Engineering tumor-homing bacteria as membrane-anchored immune checkpoint-blockade interfaces

Bacterial cancer therapies can exploit tumor tropism to localize immunomodulatory payloads, but most approaches rely on secretion or lysis-dependent release of soluble biologics that may diffuse beyond the tumor niche. Here, we engineer non-pathogenic, tumor-homing Escherichia coli strains as membrane-anchored immunotherapeutic interfaces, with individual strains displaying immune checkpoint-blocking nanobodies targeting CTLA-4 or PD-L1, either alone or in combination with a separate strain displaying murine decoy-resistant IL-18 (mDR18). By screening multiple bacterial outer-membrane proteins as scaffolds, we identified scaffold-dependent differences in display levels and target engagement, and YiaT as a functional platform for checkpoint nanobody presentation. Delivery of YiaT-displayed nanobodies in combination with OmpA-displayed mDR18 suppressed tumor growth in syngeneic mouse colon cancer and melanoma models in either local or systemic delivery. Upon systemic administration, the combined bacterial therapy preferentially accumulated in tumors, outperformed benchmark checkpoint antibody regimens combining CTLA-4 and PD-L1 under the tested conditions, and promoted tumor rejection and rechallenge resistance, without inducing broad systemic cytokine release. Further immune profiling showed that the combined treatment was associated with increased CD8⁺ and effector-memory T-cell responses in tumors and spleens. This work establishes bacterial surface display as a modular strategy for localized cancer immunotherapy.