Doctor of Philosophy, University of California San Diego (2021)
Bachelor of Science, National Cheng Kung University (2013)
Heike Daldrup-Link, Postdoctoral Faculty Sponsor
Ching-Hsin Huang, Natalie Mendez, James Wang, Tomoko Hayashi, Joi Weeks, Oscar Hernandez Echeagaray, Andrew Kummel, William C Trogler, Natalie A Gude. "United States Patent 16906933 Conjugation of tlr7 agonist to nano-materials enhances the agonistic activity", Feb 11, 2021
In Vivo Evaluation of Near-Infrared Fluorescent Probe for TIM3 Targeting in Mouse Glioma.
Molecular imaging and biology
Current checkpoint inhibitor immunotherapy strategies in glioblastoma are challenged by mechanisms of resistance including an immunosuppressive tumor microenvironment. T cell immunoglobulin domain and mucin domain 3 (TIM3) is a late-phase checkpoint receptor traditionally associated with T cell exhaustion. We apply fluorescent imaging techniques to explore feasibility of in vivo visualization of the immune state in a glioblastoma mouse model.TIM3 monoclonal antibody was conjugated to a near-infrared fluorescent dye, IRDye-800CW (800CW). The TIM3 experimental conjugate and isotype control were assessed for specificity with immunofluorescent staining and flow cytometry in murine cell lines (GL261 glioma and RAW264.7 macrophages). C57BL/6 mice with orthotopically implanted GL261 cells were imaged in vivo over 4 days after intravenous TIM3-800CW injection to assess tumor-specific uptake. Cell-specific uptake was then assessed on histologic sections.The experimental TIM3-800CW, but not its isotype control, bound to RAW264.7 macrophages in vitro. Specificity to RAW264.7 macrophages and not GL261 tumor cells was quantitatively confirmed with the corresponding clone of TIM3 on flow cytometry. In vivo fluorescence imaging of the 800CW signal was localized to the intracranial tumor and significantly higher for the TIM3-800CW cohort, relative to non-targeting isotype control, immediately after tail vein injection and for up to 48 h after injection. Resected organs of tumor bearing mice showed significantly higher uptake in the liver and spleen. TIM3-800CW was seen to co-stain with CD3 (13%), CD11b (29%), and CD206 (26%).We propose fluorescent imaging of immune cell imaging as a potential strategy for monitoring and localizing immunologically relevant foci in the setting of brain tumors. Alternative markers and target validation will further clarify the temporal relationship of immunosuppressive effector cells throughout glioma resistance.
View details for DOI 10.1007/s11307-021-01667-0
View details for PubMedID 34846678
Immunostimulatory TLR7 Agonist-Nanoparticles Together with Checkpoint Blockade for Effective Cancer Immunotherapy
2020; 3 (6)
Mono- or dual-checkpoint inhibitors for immunotherapy have changed the paradigm of cancer care; however, only a minority of patients responds to such treatment. Combining small molecule immuno-stimulators can improve treatment efficacy, but they are restricted by poor pharmacokinetics. In this study, TLR7 agonists conjugated onto silica nanoparticles showed extended drug localization after intratumoral injection. The nanoparticle-based TLR7 agonist increased immune stimulation by activating the TLR7 signaling pathway. When treating CT26 colon cancer, nanoparticle conjugated TLR7 agonists increased T cell infiltration into the tumors by > 4× and upregulated expression of the interferon γ gene compared to its unconjugated counterpart by ~2×. Toxicity assays established that the conjugated TLR7 agonist is a safe agent at the effective dose. When combined with checkpoint inhibitors that target programmed cell death protein 1 (PD-1) and cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), a 10-100× increase in immune cell migration was observed; furthermore, 100 mm3 tumors were treated and a 60% remission rate was observed including remission at contralateral non-injected tumors. The data show that nanoparticle based TLR7 agonists are safe and can potentiate the effectiveness of checkpoint inhibitors in immunotherapy resistant tumor models and promote a long-term specific memory immune function.
View details for DOI 10.1002/adtp.201900200
View details for Web of Science ID 000540590100003
View details for PubMedID 33644299
View details for PubMedCentralID PMC7904104
Indocyanine green modified silica shells for colon tumor marking
APPLIED SURFACE SCIENCE
Marking colon tumors for surgery is normally done with the use of India ink. However, non-fluorescent dyes such as India ink cannot be imaged below the tissue surface and there is evidence for physiological complications such as abscess, intestinal perforation and inconsistency of dye injection. A novel infrared marker was developed using FDA approved indocyanine green (ICG) dye and ultrathin hollow silica nanoshells (ICG/HSS). Using a positively charged amine linker, ICG was non-covalently adsorbed onto the nanoparticle surface. For ultra-thin wall 100 nm diameter silica shells, a bimodal ICG layer of < 3 nm is was formed. Conversely, for thicker walls on 2 μm diameter silica shells, the ICG layer was only bound to the outer surface and was 6 nm thick. In vitro testing of fluorescent emission showed the particles with the thinner coating were considerably more efficient, which is consistent with self-quenching reducing emission shown in the thicker ICG coatings. Ex-vivo testing showed that ICG bound to the 100 nm hollow silica shells was visible even under 1.5 cm of tissue. In vivo experiments showed that there was no diffusion of the ICG/nanoparticle marker in tissue and it remained imageable for as long as 12 days.
View details for DOI 10.1016/j.apsusc.2019.143885
View details for Web of Science ID 000502588700020
View details for PubMedID 32863496
View details for PubMedCentralID PMC7455021
- Microshell Enhanced Acoustic Adjuvants for Immunotherapy in Glioblastoma ADVANCED THERAPEUTICS 2019; 2 (10)
- Thickness and Sphericity Control of Hollow Hard Silica Shells through Iron (III) Doping: Low Threshold Ultrasound Contrast Agents ADVANCED FUNCTIONAL MATERIALS 2019; 29 (33)
Conjugation of a Small-Molecule TLR7 Agonist to Silica Nanoshells Enhances Adjuvant Activity
ACS APPLIED MATERIALS & INTERFACES
2019; 11 (30): 26637–47
Stimulation of Toll-like receptors (TLRs) and/or NOD-like receptors on immune cells initiates and directs immune responses that are essential for vaccine adjuvants. The small-molecule TLR7 agonist, imiquimod, has been approved by the FDA as an immune response modifier but is limited to topical application due to its poor pharmacokinetics that causes undesired adverse effects. Nanoparticles are increasingly used with innate immune stimulators to mitigate side effects and enhance adjuvant efficacy. In this study, a potent small-molecule TLR7 agonist, 2-methoxyethoxy-8-oxo-9-(4-carboxybenzyl)adenine (1V209), was conjugated to hollow silica nanoshells (NS). Proinflammatory cytokine (IL-6, IL-12) release by mouse bone-marrow-derived dendritic cells and human peripheral blood mononuclear cells revealed that the potency of silica nanoshells-TLR7 conjugates (NS-TLR) depends on nanoshell size and ligand coating density. Silica nanoshells of 100 nm diameter coated with a minimum of ∼6000 1V209 ligands/particle displayed 3-fold higher potency with no observed cytotoxicity when compared to an unconjugated TLR7 agonist. NS-TLR activated the TLR7-signaling pathway, triggered caspase activity, and stimulated IL-1β release, while neither unconjugated TLR7 ligands nor silica shells alone produced IL-1β. An in vivo murine immunization study, using the model antigen ovalbumin, demonstrated that NS-TLR increased antigen-specific IgG antibody induction by 1000× with a Th1-biased immune response, compared to unconjugated TLR7 agonists. The results show that the TLR7 ligand conjugated to silica nanoshells is capable of activating an inflammasome pathway to enhance both innate immune-stimulatory and adjuvant potencies of the TLR7 agonist, thereby broadening applications of innate immune stimulators.
View details for DOI 10.1021/acsami.9b08295
View details for Web of Science ID 000479020300008
View details for PubMedID 31276378