Lipid modifications provide efficient targeting of oligonucleotides to live cell membranes in a range of applications. Targeting efficiency is a function of the rate of lipid DNA insertion into the cell surface and its persistence over time. Here we show that increasing lipid hydrophobicity increases membrane persistence, but decreases the rate of membrane insertion due to the formation of nonproductive aggregates in solution. To ameliorate this effect, we split the net hydrophobicity of the membrane anchor between two complementary oligonucleotides. When prehybridized in solution, doubly anchored molecules also aggregate due to their elevated hydrophobicity. However, when added sequentially to cells, aggregation does not occur so membrane insertion is efficient. Hybridization between the two strands locks the complexes at the cell surface by increasing net hydrophobicity, increasing their total concentration and lifetime, and dramatically improving their utility in a variety of biomedical applications.
SummaryGrowth factor binding to EGFR drives conformational changes that promote homodimerization and transphosphorylation, followed by adaptor recruitment, oligomerization, and signaling through Ras. Whether specific receptor conformations and oligomerization states are necessary for efficient activation of Ras is unclear. We therefore evaluated the sufficiency of a phosphorylated EGFR dimer to activate Ras without growth factor by developing a chemical-genetic strategy to crosslink and “trap” full-length EGFR homodimers on cells. Trapped dimers become phosphorylated and recruit adaptor proteins at stoichiometry equivalent to that of EGF-stimulated receptors. Surprisingly, these phosphorylated dimers do not activate Ras, Erk, or Akt. In the absence of EGF, phosphorylated dimers do not further oligomerize or reorganize on cell membranes. These results suggest that a phosphorylated EGFR dimer loaded with core signaling adapters is not sufficient to activate Ras and that EGFR ligands contribute to conformational changes or receptor dynamics necessary for oligomerization and efficient signal propagation through the SOS-Ras-MAPK pathway.
Expansion of antibody scaffold diversity has the potential to expand the neutralizing capacity of the immune system and to generate enhanced therapeutics and probes. Systematic exploration of scaffold diversity could be facilitated with a modular and chemical scaffold for assembling proteins, such as DNA. However, such efforts require simple, modular, and site-specific methods for coupling antibody fragments or bioactive proteins to nucleic acids. To address this need, we report a modular approach for conjugating synthetic oligonucleotides to proteins with aldehyde tags at either terminus or internal loops. The resulting conjugates are assembled onto DNA-based scaffolds with low nanometer spatial resolution and can bind to live cells. Thus, this modular and site-specific conjugation strategy provides a new tool for exploring the potential of expanded scaffold diversity in immunoglobulin-based probes and therapeutics.
Checkpoint immunotherapies (CPI) have resulted in long lasting clinical responses; however, CPI treatment comes with the risk of immune-related adverse events (irAEs). Previous reports have established a correlation between irAE occurrence and better CPI patient outcomes, yet these correlations and their underlying biological mechanisms are poorly understood. To better understand the immunological mechanisms behind the correlation of irAE presentation and response, we performed immune profiling on 40 urothelial carcinoma (UC) patients receiving aPD-1 as part of the RADIOHEAD study (Lucas et al. SITC 2022, abstract #1256). A 44-marker mass cytometry panel was used to profile pretreatment and early on-treatment PBMCs to capture cell subsets and functional states across innate and adaptive immune cells. We performed analysis on gated immune cell populations and through unsupervised clustering. Group comparisons were made by adjusted Kruskal-Wallis test, whereas survival correlates were assessed through univariate Cox regression analysis. For clinical outcome analyses, we defined responders as patients who had overall survival greater than 12 months (n=26) and noted 8 patients developed irAEs. Several immune features early on-treatment were associated with overall survival. KLRG1+ naive and HLA-DR+ CD38+ central memory CD8 T cells were negatively correlated with survival, whereas Treg and TIM-3+ classical monocytes were positively associated (HR= 2.6, 3.2, 0.4, 0.05; p=0.0002, 0.00005, 0.01,0.009 respectively). Next, we classified patients into the following groups: responders with irAE (R-irAE, n=5), responders without irAE (n=21), non-responders with irAE (n=3) and non-responders without irAE (n=10). We found that several immune features were unique to R-irAE patients early on aPD-1 treatment. R-irAE patients had up to 30% more CD74+ cDCs, up to 4-fold more TIM-3+ classical monocytes, and had a greater fold decrease in CD16+ NK cells from baseline compared to other patients (p<0.005, 0.001,0.008 respectively). They also had up to 2-fold more Treg cells early on treatment, including a subset of activated Tregs marked by expression of CD38, CTLA-4, ICOS and TIGIT (p=0.05) which trended up to significance at pretreatment (p=0.075). In this study, we demonstrate that high-dimensional immune profiling by mass cytometry can detect novel blood-based biomarkers associated with clinical outcomes. Although CPI response and irAE incidence both require activation of a patient’s immune system, different mechanisms likely apply. Based on our findings, there are markers associated with responders broadly, as well as specific to R-irAE patients. We plan to validate these findings and identify additional markers and mechanisms of response and irAE with immune profiling on more RADIOHEAD indications. Citation Format: Connor P. Healy, Johanna M. Sweere, Samantha I. Liang, Natalia Sigal, Elizabeth Enrico, Li-Chun Cheng, Anastasia Lucas, Marshall A. Thompson, Diane M. Da Silva, Justin A. Jarrell, Ramji Srinivasan, Matthew H. Spitzer, Ngan Nguyen, Christine N. Spencer. Immune features of irAEs and aPD1 response in urothelial cancer patients of the RADIOHEAD study, as detected in blood by mass cytometry immune profiling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2138.
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