Deep Eutectic Solvents for Subcutaneous Delivery of Protein Therapeutics.

Proteins are among the most common therapeutics for the treatment of diabetes, autoimmune diseases, cancer, and metabolic diseases, among others. Despite their common use, current protein therapies, most of which are injectables, have several limitations. Large proteins such as monoclonal antibodies (mAbs) suffer from poor absorption after subcutaneous injections, thus forcing their administration by intravenous injections. Even small proteins such as insulin suffer from slow pharmacokinetics which poses limitations in effective management of diabetes. Here, a deep eutectic-based delivery strategy is used to offer a generalized approach for improving protein absorption after subcutaneous injections. The lead formulation enhances absorption of mAbs after subcutaneous injections by ≈200%. The same composition also improves systemic absorption of subcutaneously injected insulin faster than Humalog, the current gold-standard of rapid acting insulin. Mechanistic studies reveal that the beneficial effect of deep eutectics on subcutaneous absorption is mediated by their ability to reduce the interactions of proteins with the subcutaneous matrix, especially collagen. Studies also confirm that these deep eutectics are safe for subcutaneous injections. Deep eutectic-based formulations described here open new possibilities for subcutaneous injections of therapeutic proteins.

Use of FLOSEAL® as a scaffold and its impact on induced neural stem cell phenotype, persistence, and efficacy.

Induced neural stem cells (iNSCs) have emerged as a promising therapeutic platform for glioblastoma (GBM). iNSCs have the innate ability to home to tumor foci, making them ideal carriers for antitumor payloads. However, the in vivo persistence of iNSCs limits their therapeutic potential. We hypothesized that by encapsulating iNSCs in the FDA-approved, hemostatic matrix FLOSEAL®, we could increase their persistence and, as a result, therapeutic durability. Encapsulated iNSCs persisted for 95 days, whereas iNSCs injected into the brain parenchyma persisted only 2 weeks in mice. Two orthotopic GBM tumor models were used to test the efficacy of encapsulated iNSCs. In the GBM8 tumor model, mice that received therapeutic iNSCs encapsulated in FLOSEAL® survived 30 to 60 days longer than mice that received nonencapsulated cells. However, the U87 tumor model showed no significant differences in survival between these two groups, likely due to the more solid and dense nature of the tumor. Interestingly, the interaction of iNSCs with FLOSEAL® appears to downregulate some markers of proliferation, anti-apoptosis, migration, and therapy which could also play a role in treatment efficacy and durability. Our results demonstrate that while FLOSEAL® significantly improves iNSC persistence, this alone is insufficient to enhance therapeutic durability.

Developing Bioinspired Three-Dimensional Models of Brain Cancer to Evaluate Tumor-Homing Neural Stem Cell Therapy.

Engineered neural stem cells (NSCs) have recently emerged as a promising therapy. Acting as a tumor-homing drug-delivery system, NSCs migrate through brain tissue to seek out primary and invasive tumor foci. NSCs can deliver therapeutic agents, such as TNFα-related apoptosis-inducing ligand, directly to the tumor and suppress glioblastoma (GBM) in murine models. While the mainstays for evaluating NSC migration and efficacy have been two-dimensional chemotaxis assays and mouse models, these low-throughput and small-scale systems limit our ability to implant and track these cells for human translation. To circumvent these challenges, we developed a three-dimensional culture system using a matrix of poly-l-lactic acid 6100 microfibers suspended in agar. These bioinspired brain matrices were used to model tumor growth, NSC migration, and efficacy of NSC therapy at small and human scale. Kinetic fluorescent imaging confirmed growth of tumors in both small and human-sized bioinspired brain matrix. Tumors proliferated 50-fold and 3-fold for GBM and human metastatic breast cancer, respectively, over 7 days. We next explored the impact of tumor location on NSC migration. When NSCs were implanted 2 mm lateral from the tumor foci, NSCs colocalized with the GBM within 7 days. In models of multifocal disease, NSCs were found to colocalize with multiple tumors, preferentially migrating to tumor foci closest to the site of NSC implantation. Lastly, therapeutic NSCs were implanted at increasing distances (0, 2, 5, or 10 mm) laterally from GBM foci to investigate the effects of distance on NSC efficacy. Serial imaging showed reduced fluorescence at tumor sites, implicating GBM apoptosis across all distances. NSCs coinjected with tumor induced a near-complete response in <10 days, while NSCs implanted 10 mm laterally from the tumor induced a near-complete response by day 30. Lastly, GBM foci were established in each hemisphere of the model and control or therapeutic NSCs were implanted adjacent to tumor cells in the right hemisphere. Kinetic imaging showed that NSC therapy attenuated progression of GBM foci, while GBM cells treated with control NSC expanded rapidly over 21 days. In conclusion, we developed a new bioinspired model that supports growth of human brain cancer cells and enables rapid tracking of NSC therapy. Impact statement Tumor-homing and tumor-killing-engineered neural stem cell (NSC) therapies have shown immense promise in both preclinical and clinical trials. However, as cell therapies continue to evolve, cost-effective and high-throughput screening assays are needed to assess the proliferation, migration, and efficacy of these cells. In this study, we developed a bioinspired brain matrix for the evaluation of engineered NSCs. Importantly, this matrix is easy to fabricate, scalable, and allows for sterile real-time, noninvasive imaging using our custom bioreactor. We then utilized the bioinspired brain matrix system to answer key questions around the tumor-homing migration and efficacy of engineered NSC therapies that are challenging to address with traditional models.

Developing Bioinspired Three-Dimensional Models of Brain Cancer to Evaluate Tumor-Homing Neural Stem Cell Therapy.

Engineered neural stem cells (NSCs) have recently emerged as a promising therapy. Acting as a tumor-homing drug-delivery system, NSCs migrate through brain tissue to seek out primary and invasive tumor foci. NSCs can deliver therapeutic agents, such as TNFα-related apoptosis-inducing ligand, directly to the tumor and suppress glioblastoma (GBM) in murine models. While the mainstays for evaluating NSC migration and efficacy have been two-dimensional chemotaxis assays and mouse models, these low-throughput and small-scale systems limit our ability to implant and track these cells for human translation. To circumvent these challenges, we developed a three-dimensional culture system using a matrix of poly-l-lactic acid 6100 microfibers suspended in agar. These bioinspired brain matrices were used to model tumor growth, NSC migration, and efficacy of NSC therapy at small and human scale. Kinetic fluorescent imaging confirmed growth of tumors in both small and human-sized bioinspired brain matrix. Tumors proliferated 50-fold and 3-fold for GBM and human metastatic breast cancer, respectively, over 7 days. We next explored the impact of tumor location on NSC migration. When NSCs were implanted 2 mm lateral from the tumor foci, NSCs colocalized with the GBM within 7 days. In models of multifocal disease, NSCs were found to colocalize with multiple tumors, preferentially migrating to tumor foci closest to the site of NSC implantation. Lastly, therapeutic NSCs were implanted at increasing distances (0, 2, 5, or 10 mm) laterally from GBM foci to investigate the effects of distance on NSC efficacy. Serial imaging showed reduced fluorescence at tumor sites, implicating GBM apoptosis across all distances. NSCs coinjected with tumor induced a near-complete response in <10 days, while NSCs implanted 10 mm laterally from the tumor induced a near-complete response by day 30. Lastly, GBM foci were established in each hemisphere of the model and control or therapeutic NSCs were implanted adjacent to tumor cells in the right hemisphere. Kinetic imaging showed that NSC therapy attenuated progression of GBM foci, while GBM cells treated with control NSC expanded rapidly over 21 days. In conclusion, we developed a new bioinspired model that supports growth of human brain cancer cells and enables rapid tracking of NSC therapy. Impact statement Tumor-homing and tumor-killing-engineered neural stem cell (NSC) therapies have shown immense promise in both preclinical and clinical trials. However, as cell therapies continue to evolve, cost-effective and high-throughput screening assays are needed to assess the proliferation, migration, and efficacy of these cells. In this study, we developed a bioinspired brain matrix for the evaluation of engineered NSCs. Importantly, this matrix is easy to fabricate, scalable, and allows for sterile real-time, noninvasive imaging using our custom bioreactor. We then utilized the bioinspired brain matrix system to answer key questions around the tumor-homing migration and efficacy of engineered NSC therapies that are challenging to address with traditional models.

Author Correction: Immune and genomic correlates of response to anti-PD-1 immunotherapy in glioblastoma.

In the version of this article originally published, the graph in Extended Data Fig. 2c was a duplication of Extended Data Fig. 2b. The correct version of Extended Data Fig. 2c is now available online.

Immune and genomic correlates of response to anti-PD-1 immunotherapy in glioblastoma.

Immune checkpoint inhibitors have been successful across several tumor types; however, their efficacy has been uncommon and unpredictable in glioblastomas (GBM), where <10% of patients show long-term responses. To understand the molecular determinants of immunotherapeutic response in GBM, we longitudinally profiled 66 patients, including 17 long-term responders, during standard therapy and after treatment with PD-1 inhibitors (nivolumab or pembrolizumab). Genomic and transcriptomic analysis revealed a significant enrichment of PTEN mutations associated with immunosuppressive expression signatures in non-responders, and an enrichment of MAPK pathway alterations (PTPN11, BRAF) in responders. Responsive tumors were also associated with branched patterns of evolution from the elimination of neoepitopes as well as with differences in T cell clonal diversity and tumor microenvironment profiles. Our study shows that clinical response to anti-PD-1 immunotherapy in GBM is associated with specific molecular alterations, immune expression signatures, and immune infiltration that reflect the tumor's clonal evolution during treatment.