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In vivo molecular imaging tools hold immense potential to drive transformative breakthroughs by enabling researchers to visualize cellular and molecular interactions in real-time and/or at high resolution. These advancements will facilitate a deeper understanding of fundamental biological processes and their dysregulation in disease states. Here, we develop and characterize a self-assembling protein nanomicelle called collagen type I binding - thermoresponsive assembled protein (Col1-TRAP) that binds tightly to type I collagen in vitro with nanomolar affinity. For ex vivo visualization, Col1-TRAP is labeled with a near-infrared fluorescent dye (NIR-Col1-TRAP). Both Col1-TRAP and NIR-Col1-TRAP display approximately a 3.8-fold greater binding to type I collagen compared to TRAP when measured by surface plasmon resonance (SPR). We present a proof-of-concept study using NIR-Col1-TRAP to detect fibrotic type I collagen deposition ex vivo in the livers of mice with non-alcoholic steatohepatitis (NASH). We show that NIR-Col1-TRAP demonstrates significantly decreased plasma recirculation time as well as increased liver accumulation in the NASH mice compared to mice without disease over 4 hours. As a result, NIR-Col1-TRAP shows potential as an imaging probe for NASH with in vivo targeting performance after injection in mice. STATEMENT OF SIGNIFICANCE: Direct molecular imaging of fibrosis in NASH patients enables the diagnosis and monitoring of disease progression with greater specificity and resolution than do elastography-based methods or blood tests. In addition, protein-based imaging probes are more advantageous than alternatives due to their biodegradability and scalable biosynthesis. With the aid of computational modeling, we have designed a self-assembled protein micelle that binds to fibrillar and monomeric collagen in vitro. After the protein was labeled with near-infrared fluorescent dye, we injected the compound into mice fed on a NASH diet. NIR-Col1-TRAP clears from the serum faster in these mice compared to control mice, and accumulates significantly more in fibrotic livers.This work advances the development of targeted protein probes for in vivo fibrosis imaging., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Jin Kim Montclare reports financial support was provided by National Institutes of Health. Edward Fisher reports financial support was provided by National Institutes of Health. Ann Marie Schmidt reports financial support was provided by National Institutes of Health. Youssef Wadghiri reports financial support was provided by National Institutes of Health. Andrew Wang reports financial support was provided by National Institutes of Health. Jin Kim Montclare reports financial support was provided by National Science Foundation. Jin Kim Montclare has patent A novel collagen-binding protein micelle for the non-invasive imaging of non-alcoholic steatohepatitis (NASH) pending to Jin Kim Montclare, Andrew Wang. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Ltd.)

The development of targeted anti-cancer therapeutics offers the potential for increased efficacy of drugs and diagnostics. Utilizing modalities agnostic to tumor type, such as the hypoxic tumor microenvironment (TME), may assist in the development of universal tumor targeting agents. The hypoxia-inducible factor (HIF), in particular HIF1, plays a key role in tumor adaptation to hypoxia, and inhibiting its interaction with p300 has been shown to provide therapeutic potential. Using a multivalent assembled protein (MAP) approach based on the self-assembly of the cartilage oligomeric matrix protein coiled-coil (COMPcc) domain fused to the critical residues of the C-terminal transactivation domain (C-TAD) of the α subunit of HIF1 (HIF1α), we generate HIF1α-MAP (H-MAP). The resulting H-MAP demonstrates picomolar binding affinity to p300, the ability to downregulate hypoxia-inducible genes, and in vivo tumor targeting capability.

Triple-negative breast cancer (TNBC) lacks expressed protein targets, making therapy development challenging. Hydrogels offer a promising new route in this regard by improving the chemotherapeutic efficacy through increased solubility and sustained release. Moreover, subcutaneous hydrogel administration reduces patient burden by requiring less therapy and shorter treatment times. We recently established the design principles for the supramolecular assembly of single-domain coiled-coils into hydrogels. Using a modified computational design algorithm, we designed Q8, a hydrogel with rapid assembly for faster therapeutic hydrogel preparation. Q8 encapsulates and releases doxorubicin (Dox), enabling localized sustained release via subcutaneous injection. Remarkably, a single subcutaneous injection of Dox-laden Q8 (Q8•Dox) significantly suppresses tumors within just 1 week. This work showcases the bottom-up engineering of a fully protein-based drug delivery vehicle for improved TBNC treatment via noninvasive localized therapy.

Glioblastoma (GBM) is the most common and aggressive primary brain malignancy. Adhesion G protein-coupled receptors (aGPCRs) have attracted interest for their potential as treatment targets. Here, we show that CD97 (ADGRE5) is the most promising aGPCR target in GBM, by virtue of its de novo expression compared to healthy brain tissue. CD97 knockdown or knockout significantly reduces the tumor initiation capacity of patient-derived GBM cultures (PDGCs) in vitro and in vivo. We find that CD97 promotes glycolytic metabolism via the mitogen-activated protein kinase (MAPK) pathway, which depends on phosphorylation of its C terminus and recruitment of β-arrestin. We also demonstrate that THY1/CD90 is a likely CD97 ligand in GBM. Lastly, we show that an anti-CD97 antibody-drug conjugate selectively kills tumor cells in vitro. Our studies identify CD97 as a regulator of tumor metabolism, elucidate mechanisms of receptor activation and signaling, and provide strong scientific rationale for developing biologics to target it therapeutically in GBM., Competing Interests: Declaration of interests S.K., T.H., A. Koide, C.Y.P., D.G.P., and the NYU Grossman School of Medicine have filed a patent application titled “Anti-CD97 antibodies and antibody-drug conjugates.” D.G.P. and the NYU Grossman School of Medicine own an EU and Hong Kong patent titled “Method for treating high-grade gliomas” on the use of GPR133 as a treatment target in glioma. D.G.P. has received consultant fees from Tocagen, Synaptive Medical, Monteris, Robeaute, Guidepoint, Servier Pharmaceuticals, and Advantis. S.K. was a scientific advisory board member and received consulting fees from Black Diamond Therapeutics; is a co-founder and holds equity in Aethon Therapeutics and Revalia Bio; and has received research funding from Aethon Therapeutics, Argenx BVBA, Black Diamond Therapeutics, and Puretech Health., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Theranostic materials research is experiencing rapid growth driven by the interest in integrating both therapeutic and diagnostic modalities. These materials offer the unique capability to not only provide treatment but also track the progression of a disease. However, to create an ideal theranostic biomaterial without compromising drug encapsulation, diagnostic imaging must be optimized for improved sensitivity and spatial localization. Herein, we create a protein-engineered fluorinated coiled-coil fiber, Q2 TFL , capable of improved sensitivity to 19 F magnetic resonance spectroscopy (MRS) detection. Leveraging residue-specific noncanonical amino acid incorporation of trifluoroleucine (TFL) into the coiled-coil, Q2, which self-assembles into nanofibers, we generate Q2 TFL . We demonstrate that fluorination results in a greater increase in thermostability and 19 F magnetic resonance detection compared to the nonfluorinated parent, Q2. Q2 TFL also exhibits linear ratiometric 19 F MRS thermoresponsiveness, allowing it to act as a temperature probe. Furthermore, we explore the ability of Q2 TFL to encapsulate the anti-inflammatory small molecule, curcumin (CCM), and its impact on the coiled-coil structure. Q2 TFL also provides hyposignal contrast in 1 H MRI, echogenic signal with high-frequency ultrasound and sensitive detection by 19 F MRS in vivo illustrating fluorination of coiled-coils for supramolecular assembly and their use with 1 H MRI, 19 F MRS and high frequency ultrasound as multimodal theranostic agents., Competing Interests: The authors declare no competing financial interest., (© 2023 The Authors. Published by American Chemical Society.)

Labeled protein-based biomaterials have become a popular for various biomedical applications such as tissue-engineered, therapeutic, or diagnostic scaffolds. Labeling of protein biomaterials, including with ultrasmall super-paramagnetic iron oxide (USPIO) nanoparticles, has enabled a wide variety of imaging techniques. These USPIO-based biomaterials are widely studied in magnetic resonance imaging (MRI), thermotherapy, and magnetically-driven drug delivery which provide a method for direct and non-invasive monitoring of implants or drug delivery agents. Where most developments have been made using polymers or collagen hydrogels, shown here is the use of a rationally designed protein as the building block for a meso-scale fiber. While USPIOs have been chemically conjugated to antibodies, glycoproteins, and tissue-engineered scaffolds for targeting or improved biocompatibility and stability, these constructs have predominantly served as diagnostic agents and often involve harsh conditions for USPIO synthesis. Here, we present an engineered protein-iron oxide hybrid material comprised of an azide-functionalized coiled-coil protein with small molecule binding capacity conjugated via bioorthogonal azide-alkyne cycloaddition to an alkyne-bearing iron oxide templating peptide, CMms6, for USPIO biomineralization under mild conditions. The coiled-coil protein, dubbed Q, has been previously shown to form nanofibers and, upon small molecule binding, further assembles into mesofibers via encapsulation and aggregation. The resulting hybrid material is capable of doxorubicin encapsulation as well as sensitive T 2 *-weighted MRI darkening for strong imaging capability that is uniquely derived from a coiled-coil protein., Competing Interests: The authors declare no competing financial interests.

Background: Preclinical studies indicate that cannabidiol (CBD), the primary nonaddictive component of cannabis, has a wide range of reported pharmacological effects such as analgesic and anxiolytic actions; however, the exact mechanisms of action for these effects have not been examined in chronic osteoarthritis (OA). Similar to other chronic pain syndromes, OA pain can have a significant affective component characterized by mood changes. Serotonin (5-HT) is a neurotransmitter implicated in pain, depression, and anxiety. Pain is often in comorbidity with mood and anxiety disorders in patients with OA. Since primary actions of CBD are analgesic and anxiolytic, in this first in vivo positron emission tomography (PET) imaging study, we investigate the interaction of CBD with serotonin 5-HT 1A receptor via a combination of in vivo neuroimaging and behavioral studies in a well-validated OA animal model., Methods: The first aim of this study was to evaluate the target involvement, including the evaluation of modulation by acute administration of CBD, or a specific target antagonist/agonist intervention, in control animals. The brain 5-HT 1A activity/availability was assessed via in vivo dynamic PET imaging (up to 60 min) using a selective 5-HT 1A radioligand ([ 18 F]MeFWAY). Tracer bindings of 17 ROIs were evaluated based on averaged SUVR values over the last 10 min using CB as the reference region. We subsequently examined the neurochemical and behavioral alterations in OA animals (induction with monosodium iodoacetate (MIA) injection), as compared to control animals, via neuroimaging and behavioral assessment. Further, we examined the effects of repeated low-dose CBD treatment on mechanical allodynia (von Frey tests) and anxiety-like (light/dark box tests, L/D), depressive-like (forced swim tests, FST) behaviors in OA animals, as compared to after vehicle treatment., Results: The tracer binding was significantly reduced in control animals after an acute dose of CBD administered intravenously (1.0 mg/kg, i.v.), as compared to that for baseline. This binding specificity to 5-HT 1A was further confirmed by a similar reduction of tracer binding when a specific 5-HT 1A antagonist WAY1006235 was used (0.3 mg/kg, i.v.). Mice subjected to the MIA-induced OA for 13-20 days showed a decreased 5-HT 1A tracer binding (25% to 41%), consistent with the notion that 5-HT 1A plays a role in the modulation of pain in OA. Repeated treatment with CBD administered subcutaneously (5 mg/kg/day, s.c., for 16 days after OA induction) increased 5-HT 1A tracer binding, while no significant improvement was observed after vehicle. A trend of increased anxiety or depressive-like behavior in the light/dark box or forced swim tests after OA induction, and a decrease in those behaviors after repeated low-dose CBD treatment, are consistent with the anxiolytic action of CBD through 5HT 1A receptor activation. There appeared to be a sex difference: females seem to be less responsive at the baseline towards pain stimuli, while being more sensitive to CBD treatment., Conclusion: This first in vivo PET imaging study in an OA animal model has provided evidence for the interaction of CBD with the serotonin 5-HT 1A receptor. Behavioral studies with more pharmacological interventions to support the target involvement are needed to further confirm these critical findings., Competing Interests: Competing interests: The authors declare that they have no competing interests.