Periodic Reporting for period 2 - PB_LC (Clinical readiness of a live biotherapeutic for treatment of Non-Small Cell Lung Cancer (NSCLC))
Período documentado: 2024-01-01 hasta 2025-08-31
Lung cancer remains one of the most pressing health challenges worldwide, ranking as the second most frequently diagnosed cancer and the leading cause of cancer-related deaths. Non-small cell lung cancer (NSCLC) represents about 85% of cases, and most patients are diagnosed at advanced stages, when curative treatment options are limited and survival outcomes remain poor.
Immune checkpoint inhibitors (ICIs) have transformed cancer therapy by reactivating the immune system to attack tumors. However, many patients fail to respond or develop resistance because their tumors remain “immunologically cold” — poorly infiltrated by immune cells and thus less visible to immune attack. This resistance limits the long-term success of immunotherapy and highlights the need for complementary strategies to make “cold” tumors “hot”.
The PB_LC project was designed to address this challenge by developing a live biotherapeutic product based on Mycoplasma pneumoniae, a bacterium naturally adapted to the lung environment. Using synthetic biology, Pulmobiotics engineered this organism to secrete immune-stimulating proteins within the tumor microenvironment. Administered by inhalation, PB_LC was intended to transiently colonize the lung and locally trigger immune activation, thereby enhancing the efficacy of existing immunotherapies.
Following early progress, the project’s objectives were refined to identify and develop a strain with single-agent therapeutic activity, establishing clear intrinsic efficacy before combining with ICIs. The overarching goal was to demonstrate the feasibility of using a live bacterium as a programmable drug-delivery system for cancer therapy, paving the way for novel microbial medicines that could complement current immunotherapies and improve outcomes for patients with advanced lung cancer.
Immune checkpoint inhibitors (ICIs) have transformed cancer therapy by reactivating the immune system to attack tumors. However, many patients fail to respond or develop resistance because their tumors remain “immunologically cold” — poorly infiltrated by immune cells and thus less visible to immune attack. This resistance limits the long-term success of immunotherapy and highlights the need for complementary strategies to make “cold” tumors “hot”.
The PB_LC project was designed to address this challenge by developing a live biotherapeutic product based on Mycoplasma pneumoniae, a bacterium naturally adapted to the lung environment. Using synthetic biology, Pulmobiotics engineered this organism to secrete immune-stimulating proteins within the tumor microenvironment. Administered by inhalation, PB_LC was intended to transiently colonize the lung and locally trigger immune activation, thereby enhancing the efficacy of existing immunotherapies.
Following early progress, the project’s objectives were refined to identify and develop a strain with single-agent therapeutic activity, establishing clear intrinsic efficacy before combining with ICIs. The overarching goal was to demonstrate the feasibility of using a live bacterium as a programmable drug-delivery system for cancer therapy, paving the way for novel microbial medicines that could complement current immunotherapies and improve outcomes for patients with advanced lung cancer.
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.
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.
PB_LC advanced the state of the art in both cancer immunotherapy and synthetic biology by showing that a rationally engineered, lung-adapted bacterium can serve as a programmable live biotherapeutic to modulate the tumor microenvironment. Unlike previous bacterial therapies, PB_LC introduced a lung-specific microorganism designed to transiently colonize tumors and locally deliver immune-stimulatory factors where they are most needed.
This strategy directly addresses a key limitation of current immunotherapies — the lack of immune activation in “cold” tumors. By promoting local inflammation and T-cell infiltration, PB_LC could enhance the responsiveness of resistant tumors to ICIs and potentially other treatments.
Technological achievements beyond the current state of the art include:
Establishment of robust ICI-resistant lung cancer models for microbial therapy evaluation.
Generation of engineered M. pneumoniae strains expressing multiple immunomodulatory payloads with controlled activity and transient persistence.
Development of scalable fermentation, analytical and formulation processes compatible with pharmaceutical standards, including optimized growth conditions and stability testing workflows.
An additional innovation was the groundwork for aerosol delivery of live biotherapeutics. Further work on nebulizer selection, aerosol characterization, and in-vivo deposition performance will be needed to ensure efficient and reproducible lung administration.
Future steps beyond the project include expanded preclinical validation, formal toxicology studies, optimization of delivery systems, process scale-up under GMP, and establishing partnerships to advance this emerging therapeutic class toward clinical translation.
Although the project concluded earlier than planned, it generated valuable scientific knowledge, technological capabilities, and regulatory insights, establishing a solid foundation for future development of microbial immunotherapies for cancer.
This strategy directly addresses a key limitation of current immunotherapies — the lack of immune activation in “cold” tumors. By promoting local inflammation and T-cell infiltration, PB_LC could enhance the responsiveness of resistant tumors to ICIs and potentially other treatments.
Technological achievements beyond the current state of the art include:
Establishment of robust ICI-resistant lung cancer models for microbial therapy evaluation.
Generation of engineered M. pneumoniae strains expressing multiple immunomodulatory payloads with controlled activity and transient persistence.
Development of scalable fermentation, analytical and formulation processes compatible with pharmaceutical standards, including optimized growth conditions and stability testing workflows.
An additional innovation was the groundwork for aerosol delivery of live biotherapeutics. Further work on nebulizer selection, aerosol characterization, and in-vivo deposition performance will be needed to ensure efficient and reproducible lung administration.
Future steps beyond the project include expanded preclinical validation, formal toxicology studies, optimization of delivery systems, process scale-up under GMP, and establishing partnerships to advance this emerging therapeutic class toward clinical translation.
Although the project concluded earlier than planned, it generated valuable scientific knowledge, technological capabilities, and regulatory insights, establishing a solid foundation for future development of microbial immunotherapies for cancer.