Systemic Checkpoint Blockade by PD-L1 Single-Chain Antibody Confers Potent Antitumor Immunity and Long-term Survival.

Immune checkpoint inhibitors (ICI) are promising in adjuvant settings for solid tumors and hematologic malignancies. They are currently used in the treatment as mAbs in high concentrations, raising concerns of toxicity and adverse side effects. Among various checkpoint molecules, targeting the programmed cell death protein-1 (PD-1)-programmed death-ligand 1 (PD-L1) axis has garnered more clinical utility than others have. To develop a physiologically relevant and systemically stable level of ICIs from a one-time application by genetic antibody engineering, we endeavored using a nonpathogenic, replication-deficient recombinant adeno-associated vector (rAAV) expressing single-chain variable fragments (scFv) of PD-L1 antibody and tested in syngeneic mouse therapy models of MC38 colorectal and EMT6 breast tumors. Results of this study indicated a significant protection against PD-L1-mediated inhibition of CD8+ T-cell function, against the growth of primary and secondary tumors, and durable antitumor CTLs activity by adoptive CD8+ T-cell transfer. Stable maintenance of PD-L1 scFv in vivo resulted in an increase in PD-1- CD8+ T cells and a concomitant decrease in regulatory T cells, M2 macrophages, and myeloid-derived suppressor cells in the tumor microenvironment. Overall, these data demonstrate the potential of rAAV-PD-L1-scFv as an alternative to mAb targeting of PD-L1 for tumor therapy.

RANKL-Targeted Combination Therapy with Osteoprotegerin Variant Devoid of TRAIL Binding Exerts Biphasic Effects on Skeletal Remodeling and Antitumor Immunity.

Complexities in treating breast cancer with bone metastasis are enhanced by a vicious protumorigenic pathology, involving a shift in skeletal homeostasis toward aggressive osteoclast activity and polarization of immune cells supporting tumor growth and immunosuppression. Recent studies signify the role of receptor activator of NF-κB ligand (RANKL) beyond skeletal pathology in breast cancer, including tumor growth and immunosuppression. By using an osteoprotegerin (OPG) variant, which we developed recently through protein engineering to uncouple TNF-related apoptosis-inducing ligand (TRAIL) binding, this study established the potential of a cell-based OPGY49R therapy for both bone damage and immunosuppression in an immunocompetent mouse model of orthotopic and metastatic breast cancers. In combination with agonistic death receptor (DR5) activation, the OPGY49R therapy significantly increased both bone remolding and long-term antitumor immunity, protecting mice from breast cancer relapse and osteolytic pathology. With limitations, cost, and toxicity issues associated with the use of denosumab, bisphosphonates, and chemotherapy for bone metastatic disease, use of OPGY49R combination could offer a viable alternate therapeutic approach.

Recombinant AAV-CEA Tumor Vaccine in Combination with an Immune Adjuvant Breaks Tolerance and Provides Protective Immunity.

Carcinoembryonic antigen (CEA) is a human glycoprotein involved in cellular adhesion and expressed during human fetal development. Although expression of CEA largely ceases prior to birth, several human epithelial cancers, including colorectal, gastric, squamous esophageal, and breast carcinomas have been known to overexpress CEA, suggesting its potential as an immunotherapeutic target. Using a transgenic mouse model constitutively expressing human CEA in a spatiotemporal manner as a self-protein and a syngeneic mouse colon cancer cell line, MC38-CEA, overexpressing CEA, we tested the potential of a novel genetic immunotherapy approach against CEA-expressing tumors, using recombinant adeno-associated virus vector encoding CEA (rAAV-CEA) and appropriately timed immune adjuvant application. Results of the study demonstrated breaking of immune tolerance for CEA with this vaccine regimen and an anti-tumor response, resulting in tumor-free survival. Furthermore, tumor challenge of CEA-vaccinated mice with parental MC38 cells not expressing CEA did not result in protection from tumor development, confirming that the protection against tumor development is CEA specific. The study illustrates the feasibility of utilizing rAAV vectors in combination with an immunostimulatory adjuvant to break tolerance to weakly immunogenic self-antigens and for an anti-tumor response.

Characterization of immune cell subtypes in three commonly used mouse strains reveals gender and strain-specific variations.

The lack of consensus on bone marrow (BM) and splenic immune cell profiles in preclinical mouse strains complicates comparative analysis across different studies. Although studies have documented relative distribution of immune cells from peripheral blood in mice, similar studies for BM and spleen from naïve mice are lacking. In an effort to establish strain- and gender-specific benchmarks for distribution of various immune cell subtypes in these organs, we performed immunophenotypic analysis of BM cells and splenocytes from both genders of three commonly used murine strains (C57BL/6NCr, 129/SvHsd, and BALB/cAnNCr). Total neutrophils and splenic macrophages were significantly higher in C57BL/6NCr, whereas total B cells were lower. Within C57BL/6NCr female mice, BM B cells were elevated with respect to the males whereas splenic mDCs and splenic neutrophils were reduced. Within BALB/cAnNCr male mice, BM CD4+ Tregs were elevated with respect to the other strains. Furthermore, in male BALB/cAnNCr mice, NK cells were elevated with respect to the other strains in both BM and spleen. Splenic CD4+ Tregs and splenic CD8+ T cells were reduced in male BALB/c mice in comparison to female mice. Bone marrow CD4+ T cells and mDCs were significantly increased in 129/SvHsd whereas splenic CD8+ T cells were reduced. In general, males exhibited higher immature myeloid cells, macrophages, and NK cells. To our knowledge, this study provides a first attempt to systematically establish organ-specific benchmarks on immune cells in studies involving these mouse strains.

Location of tumor affects local and distant immune cell type and number.

INTRODUCTION: Tumors comprise heterogeneous populations of cells, including immune infiltrates that polarize during growth and metastasis. Our preclinical studies on breast cancer (BCa) identified functional differences in myeloid-derived suppressor cells based on tumor microenvironment (TME), prompting variations in host immune response to tumor growth, and dissemination based on tissue type. METHODS: In order to understand if such variations existed among other immune cells, and if such alteration occurs in response to tumor growth at the primary site or due to bone dissemination, we characterized immune cells, examining localized growth and in the tibia. In addition, immune cells from the spleen were examined from animals of both tumor locations by flow cytometry. RESULTS: The study demonstrates that location of tumor, and not simply the tumor itself, has a definitive role in regulating immune effectors. Among all immune cells characterized, macrophages were decreased and myeloid dendritic cell were increased in both tumor locations. This difference was more evident in subcutaneous tumors. Additionally, spleens from mice with subcutaneous tumors contained greater increases in both macrophages and myeloid dendritic cells than in mice with bone tumors. Furthermore, in subcutaneous tumors there was an increase in CD4+ and CD8+ T-cell numbers, which was also observed in their spleens. CONCLUSIONS: These data indicate that alterations in tumor-reactive immune cells are more pronounced at the primary site, and exert a similar change at the major secondary lymphoid organ than in the bone TME. These findings could provide translational insight into designing therapeutic strategies that account for location of metastatic foci.