Presenter:
Alan Long
Glioblastoma is a devastating type of brain tumor, affecting over 13000 Americans every year. With a 5-year survival rate of only 5%, there is an urgent need for effective therapies. Glioblastoma is an immunologically cold tumor, characterized by an immunosuppressed tumor microenvironment (TME) with minimal infiltration of immune cells, which are often exhausted. We aim to generate personalized therapies by expanding tumor-specific, non- exhausted Tumor-Infiltrating Lymphocytes (TILs) derived from patient samples. Past attempts have faced the problem that these TILs may still become exhausted when reintroduced into the immunosuppressive TME. Thus, additional research is necessary to identify TIL gene targets that can be modified to produce a more resilient phenotype. We have generated TILs using a Rapid Expansion Protocol (REP) and preREP culture protocols. We then used flow cytometry to assess changes in immune cell populations between 2 variations of preREP protocols (one standard and one with a bi-specific antibody blocking 4-1BB and PD- L1) and the same samples after also undergoing the standard REP. For both protocols, our results demonstrated a marked increase in the proportion of TILs after REP, indicating favorable TIL growth, while preventing proliferation of residual tumor cells in the sample. Additionally, we found that both protocols promote proliferation of classical CD4+ T cells, and curiously, the pre-REP with the antibody also encouraged growth of innate immune cells, like Natural Killer (NK) T cells and gamma delta-T cells. These results provide an important baseline for understanding the specific cell types selected for in the REP stage, as well as the pre-REP protocols with and without the 4-1BB:PDL1 antibody. Future experiments include CRISPR screens and infiltration assays that could help develop a more effective TIL therapy for glioblastoma. Discovery of Intracellular Biased Allosteric Ligands Targeting β2-Adrenergic Receptors through In Vitro Screening
Abstract profile. Full document pending author claim.
Authors:
Presenter: Alan Long
Date Created:
Not specified
Course Title:
Professor:
Not specified
About Paper:
G protein-coupled receptors (GPCRs) represent the most pharmacologically exploited family of membrane proteins with over one third of FDA approved drugs targeting them. Their functional versatility enables them to receive diverse extracellular stimuli and transduce those signals into intracellular responses through G protein coupling and Beta-arrestin recruitment. In asthma, Beta2-Adrenergic Receptor, a specific GPCR subtype, mediated G protein signaling promotes bronchial airway smooth muscle relaxation while Beta-arrestin recruitment promotes inflammation. In heart failure these roles are reversed: Beta-arrestin recruitment is cardioprotective while G protein coupling exacerbates cardiac stress. Therefore, an ideal drug candidate would be one that biases the pathway that promotes therapeutic effects while downregulating the pathway that initiates adverse effects. Current treatments for both conditions act on the orthosteric sites of the receptor, where endogenous ligands bind, which induce balanced signaling and greater off-target effects that lead to undesirable side effects. Recognizing these pharmacological constraints, recent efforts have turned toward allosteric sites, topographically distinct domains that minimize off-target effects and offer opportunities to fine tune GPCR conformation and signaling pathway. We are focusing on identifying biased small molecule allosteric modulators (AMs) as improved asthma and heart failure therapeutics. To achieve this, a range of pharmacological characterization assays have been implemented to evaluate the receptor binding features and behavior of two dozen analogs of Compound-6, a balanced positive AM of the Beta2-Adrenergic Receptor discovered by the Lefkowitz Lab. Particularly during this summer, we have utilized in vitro assays, including 3H- fenoterol binding assay, to screen for potential biased molecules and obtain preliminary structure activity relationship (SAR) information. Beta-arrestin1, a key component for these assays, was expressed and purified to achieve high quality and yield. With this SAR information, we aim to ultimately develop such molecules as safer and more efficacious treatment for asthma and heart failure with minimal side effects.
Source:
Duke University / 2025
Topics:
No topics listed
Co-authors:
Presenter: Alan Long