Quiñones‐Hinojosa, Alfredo

Abstract Despite its success in treating blood cancers, chimeric antigen receptor (CAR) T-cell therapy has not yielded anticipated clinical outcomes in patients with solid tumors. This discrepancy is primarily attributed to the lack of dependable antigens and the immunosuppressive nature of the tumor microenvironment (TME). A pivotal evasion mechanism employed by tumor to counter antitumor immunity involves the upregulation of programmed death ligand 1 (PD-L1), which interacts with PD1 on T cells, deactivating effector T cells. A variety of immunosuppressive cells within the TME employ similar mechanisms to inhibit T-cell functions. These findings provide a rationale for targeting PD-L1 in both tumors and the immunosuppressive cells. Using a humanized anti-PD-L1 monoclonal antibody, we engineered a second-generation PD-L1 CAR, namely MC9999. MC9999 CAR T-cells exhibited antigen-specific cytotoxicity in vitro and elicited potent in vivo antitumor effects against PD-L1-expressing MDA-MD-232 breast cancer cells. The effectiveness of MC9999 CAR T-cells was confirmed across three other solid tumor models: non-small cell lung cancer, melanoma and glioblastoma (GBM). Notably, intravenous administration of MC9999 CAR T-cells eradicated intracranially established LN229 GBM tumors, suggesting the potential to penetrate the blood-brain barrier. In addition to targeting tumor cells, MC9999 CAR T-cells exhibited cytotoxicity against three immunosuppressive cell models, including HMC3 microglia cells, M2 macrophages, and importantly, primary tumor-associated macrophages (TAMs) isolated from GBM. Furthermore, we engineered patient-derived MC9999 CAR T-cells and demonstrated their effectiveness against two primary tumor cell lines derived from GBM tumors and HMC3 cells. Collectively, these proof-of-principle study findings substantiate the viability of targeting PD-L1 against both tumors and their immunosuppressive microenvironment. This prototype MC9999 CAR lays the groundwork for further development of clinically applicable CAR T-cell therapy against solid tumors. Citation Format: Yaqing Qie, Emiliano Sanchez Garavito, Maria J. Ulloa Navas, Tanya Hundal, Alfredo Quinones-Hinojosa, Hong Qin, Yan Luo. Weaponizing CAR T-cells to attack PD-L1 presenting cells in solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB071.

Abstract Glioblastoma remains as one of the deadliest malignancies with most cases likely to recur. Recent work has focused on characterizing not only glioma cells, but the tumor microenvironment via single-cell and spatial transcriptomic technologies. This has improved our understanding of the high heterogeneity of this disease and described potential tumor domains. However, there is still a need to further understand the changes that occur between the primary and the recurrent lesion. We propose that a spatial, multi-omic characterization of matched primary and recurrent glioblastoma can yield insights into recurrence mechanisms occurring throughout the tumor microenvironment. We collected 22 IDH-WT glioblastoma samples from 12 patients (11 primary, 11 recurrent, 10 matched pairs) from the Mayo Clinic Neuroscience Biobank. We performed single-nucleus RNA sequencing (15 samples, 10 patients, 5 matched pairs) and proteomics (16 samples, 6 matched pairs) from fresh-frozen tissue, as well as spatial transcriptomics with 10x Visium (14 samples, 8 patients, 6 matched pairs) and 10x Xenium in-situ molecular imaging (6 samples, 2 matched pairs). Using spatial transcriptomics data, we identified regional subsets present in different proportions and distributions between primary and recurrent glioblastoma. These clusters were characterized with gene set and gene ontology analysis, identifying regions enriched with known glioma subtypes and cellular activity. Spatial-based cluster genes were consistent with significantly differentially expressed proteins. We used single-nucleus RNAseq and in-situ molecular imaging data from matched tissue samples to further interrogate relevant cell populations and location in the tumor micro-environment. We identified regions with enriched extra cellular matrix interaction and remodeling signatures to be highly expressed in recurrent glioblastoma. Primary glioblastoma was enriched with oligodendrocyte-like regions with high cell communication and extracellular vesicle signatures. This study will further our understanding of the changes in the mechanisms at play in the complex ecosystem of glioblastoma as it recurs. Citation Format: Jean R. Clemenceau, Paola Suarez-Meade, Alfredo Quiñones-Hinojosa, Tae Hyun Hwang. Spatial and multi-omic interrogation of the primary and recurrent glioblastoma microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5482.

Abstract Glioblastoma multiforme (GBM) is a highly lethal human cancer thought to originate from a self-renewing and therapeutically-resistant population of glioblastoma stem cells (GSCs). The intrinsic mechanisms enacted by GSCs during 3D tumor formation, however, remain unclear, especially in the stages prior to angiogenic/immunological infiltration. In this study, we performed a deep characterization of the genetic, immune, and metabolic profiles of GBM organoids from several patient-derived GSCs (GBMO). Despite being devoid of immune cells, transcriptomic analysis across GBMO revealed a surprising immune-like molecular program, enriched in cytokine, antigen presentation and processing, T-cell receptor inhibitors, and interferon genes. We find two important cell populations thought to drive GBM progression, Special AT-rich sequence-binding protein 2 (SATB2+) and homeodomain-only protein homeobox (HOPX+) progenitors, contribute to this immune landscape in GBMO and GBM in vivo. These progenitors, but not other cell types in GBMO, are resistant to conventional GBM therapies, temozolomide and irradiation. Our work defines a novel intrinsic immune-like landscape in GBMO driven, in part, by SATB2+ and HOPX+ progenitors and deepens our understanding of the intrinsic mechanisms utilized by GSCs in early GBM formation.