During the second reporting period, the project focused on validating a live biotherapeutic strain with single-agent activity in models of ICI-resistant lung cancer.
Pulmobiotics conducted new in vivo efficacy studies using both subcutaneous and orthotopic lung tumor models that better represent advanced NSCLC. Two complementary mouse models were established: B16F10-OVA foci, to evaluate tumor burden and immune infiltration, and KPB6_BLI, to assess longitudinal tumor growth and therapeutic response in the lung. These provided a robust framework to evaluate engineered M. pneumoniae strains expressing different immune-stimulatory payloads.
More than ten new strains were tested. Two candidates emerged: C209, which improved survival when combined with anti-PD-L1 therapy, and PBLC_P1b, which showed measurable single-agent anti-tumor activity in the KPB6_BLI model. These findings supported the project’s shift toward developing a stand-alone therapeutic candidate with intrinsic efficacy.
Immune-profiling studies revealed that active strains increased T-cell (especially CD4+) and dendritic-cell infiltration into tumors and promoted Th1-type cytokines such as interferon-gamma and interleukin-12, confirming local immune activation.
Biodistribution and toxicology studies showed that the engineered strains were rapidly cleared from the lungs within days and did not spread to other organs. Histopathology indicated only mild, transient inflammation comparable to controls, supporting a favorable safety profile.
Significant advances were also made in manufacturing and formulation development. Scalable upstream processes were established in bioreactors, and analytical methods such as Quantom TX™ and dynamic light scattering (DLS) were implemented to support future process validation. Two formulation approaches — lyophilization and spray-drying — were optimized, both maintaining bacterial viability for several months, with lyophilization offering slightly better long-term stability. A GMP-compatible, animal-free medium was identified, ensuring robust strain performance for large-scale production.
The formal candidate strain was nominated in August 2025, shortly before the decision to terminate the project. Although this prevented initiation of the planned regulatory and GMP activities, the scientific and technological advances achieved in RP2 provide a strong foundation for any future continuation of the PB_LC program.
