Proteolysis-targeting chimeras (PROTACs) and degraders have been developed against the hRpn13 fragment hRpn13Pru that is present in various cancer types. Testing the performance of these hRpn13Pru-targeting compounds in pharmacokinetic and efficacy studies has been stymied, however, by their poor solubility. Here, we develop a rapid and cost-effective platform to Screen and Characterize small molecule Nanosuspensions (SCaN). We discovered that the hRpn13Pru degrader XL44 adopts a crystalline state, preventing its bioavailability. The first phase of SCaN screens vehicles to identify lead XL44 nanosuspension formulations based on particle size consistency, including in biorelevant media, while the second phase evaluates stability over time. The lead nanosuspensions are then advanced to the third phase of SCaN to assess their physical and colloidal stability. This pipeline allows the formulation of poorly soluble compounds for single-dose pharmacokinetic and pilot multidose tumor mouse studies. We additionally analyzed our XL44 formulation morphologically by atomic force microscopy to find that the XL44 nanoparticles are predominantly globular, with a small population of rod-like particles. Using the optimal nanosuspension determined by SCaN, XL44 slowed tumor growth in a myeloma xenograft model at 35% inhibition with a 48-72 mg/kg treatment regimen. This case study is the first in vivo demonstration that hRpn13Pru-targeting degraders can inhibit tumor growth, and the efficacy shown here motivates the development of more potent hRpn13Pru degraders. Broadly, our SCaN platform is designed for poorly soluble drug candidates to allow for pilot in vivo testing.

Esophageal squamous cell carcinoma (ESCC) is one of the deadliest cancers worldwide due to its aggressive nature and lack of knowledge of underlying oncogenic drivers, limiting treatment options. Approximately 60% of ESCC cases have amplification/overexpression of MAP3K13, which encodes the kinase LZK. Here, we found that MAP3K13-amplified ESCC exhibit therapeutic dependency on LZK, and that small-molecule inhibition of its catalytic function decreased the viability of ESCC cells with amplified MAP3K13. Inhibition of LZK suppressed tumor growth in MAP3K13-amplified ESCC patient-derived xenograft mice treated orally with a newly described LZK inhibitor. We discovered that LZK is required to sustain AKT activation in ESCC and HNSCC, where depletion, degradation, or treatment with the LZK inhibitor GNE-3511 or an improved inhibitor suppressed AKT activation. AKT activation could be rescued by expression of an LZK drug-resistant mutant, indicating suppression was specific to LZK. AKT and LZK co-localized and interacted in cells, and LZK enhanced AKT phosphorylation at S473 and T450 in vitro. This pattern of AKT phosphorylation reflected a non-catalytic scaffold function of LZK, as LZK inhibitors did not suppress AKT activation and a kinase-dead LZK mutant promoted AKT activation. AKT inhibitors reduced LZK-induced activation of AKT, indicating LZK facilitates AKT autophosphorylation. Furthermore, GNE-3511 inhibited the interaction of LZK with AKT in co-immunoprecipitation experiments. Molecular modeling supported a mechanism whereby LZK binds and dislodges AKT's PH domain to promote AKT autophosphorylation. Our findings demonstrate that MAP3K13-amplified tumors are dependent on LZK-mediated AKT scaffolding, supporting LZK inhibition as a therapeutic strategy in ESCC and HNSCC.

Synaptic vesicle glycoprotein 2A (SV2A) is a 12-pass transmembrane protein expressed in presynaptic vesicles. Positron emission tomography (PET) imaging of SV2A provides an in vivo measure of synaptic density and has broad applications in the study of neuropsychiatric and neurodegenerative diseases. A fluorine-18-labeled PET tracer targeting SV2A, [18F]SynVesT-1, was originally developed using a copper-mediated fluorination approach that requires initial azeotropic drying of [18F]fluoride with anhydrous acetonitrile. We previously established an efficient radiolabeling strategy in which fluorine-18 retained on an anion-exchange cartridge is eluted as 4-dimethylaminopyridinium [18F]fluoride (DMAPH·[18F]F) by passing a solution of 4-dimethylaminopyridinium trifluoromethanesulfonate (DMAPH·OTf) in dimethylacetamide (DMA), which was directly used in copper-mediated fluorination of various substrates without the need for azeotropic drying. Building on this strategy, we developed a fully automated and reproducible method for the synthesis of [18F]SynVesT-1 using the Trasis All-in-One (AIO) module. The total synthesis time was 60 min, affording a superior overall decay-corrected radiochemical yield (30–37% vs. 20.6 ± 1.2%) while requiring a reduced amount of precursor (2 mg vs. 5 mg) with a radiochemical purity greater than 98%.

Introduction:Lung cancer in never-smokers is a growing, biologically distinct entity lacking non-invasive markers. Established urinary markers-creatine riboside (CR) and N-acetylneuraminic acid (NANA)-report tumor-intrinsic metabolism, not carcinogen processing. We investigated 27-nor-5β-cholestane-3α,7α,12α,24R,25S-pentol glucuronide (CPG), a bile-acid glucuronide linked to aryl-hydrocarbon-receptor (AhR)/CYP xenobiotic metabolism. Methods:Urinary CPG was quantified by UPLC-tandem mass spectrometry in an exploratory (NCI-Maryland; n=846) and validation (Colorado; n=505) cohort of non-small-cell lung cancer cases and frequency-matched controls. Associations with case status, smoking stratum, survival, and discrimination were assessed, using tumor RNA sequencing (n=83) and gene-set enrichment analysis (GSEA). Results:Urinary CPG was higher in cases than controls in both cohorts (P<0.0001). In never-smokers, cases exceeded smoking-matched controls (P<0.001 and P<0.0001), indicating elevation independent of tobacco exposure. After mutual adjustment for CR and NANA, CPG remained independently associated with case status (exploratory OR 1.58, 95% CI 1.15-2.16; validation OR 3.92, 95% CI 2.47- 6.29), with a modest gain in discrimination. High CPG identified never-smokers with worse survival in both cohorts (P<0.001 and P=0.04), remaining significant after multivariable adjustment only in the exploratory cohort. GSEA showed AhR/CYP xenobiotic and Nrf2 oxidative-stress enrichment in high-CPG tumors; the CPG aglycone carried disease-specific 24R,25S stereochemistry. Conclusions:Urinary CPG was associated with NSCLC in two retrospective case-control cohorts, including in a smoking-matched never-smoker comparison. High CPG also identified never-smokers with worse survival, remaining independently prognostic after adjustment in the exploratory cohort. Tumor expression does not establish tissue of origin. Prospective validation against CR and NANA is required.

Lipid nanoparticles (LNPs) have emerged as a promising nonviral nucleic acid delivery platform for clinical use. To expand LNPs as a treatment option in nonhepatic-based diseases, LNPs surface coated with targeting moieties produce a precise modular delivery method that can bind to and be internalized by specific receptors expressed on target cells. This study showcases the tetrazine-trans-cyclooctene inverse electron-demand Diels-Alder click reaction and directly compares its performance with the widely employed thiol-maleimide conjugation. We also compare direct mixing and micelle mixing insertion methods under different conditions to determine the optimal formulation to produce targeted LNPs. For thiol-maleimide chemistry, monoclonal antibody cetuximab was modified with N-succinimidyl S-acetylthioacetate, followed by reaction with either 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethylene glycol)-2000] (DSPE-PEG2000-maleimide) directly or in preformed DSPE-PEG2000-maleimide: 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG2000) micelles. For clickable tetrazine chemistry, cetuximab was modified with 2,5-dioxo-1-pyrrolidinyl 5-[4-(1,2,4,5-tetrazin-3-yl)benzylamino]-5-oxopentanoate, followed by reaction with either 1,2-Distearoyl-sn-glycero-3-PE-polyethylene glycol-2000- trans-cyclooctene (DSPE-PEG2000-TCO) directly or preformed DSPE-PEG2000-TCO:DMG-PEG2000 micelles. LNPs were prepared by mixing lipids in ethanol and mRNA (10 mM citrate buffer, pH 3.5) at a ratio of 3:1 (v/v). Insertion was carried out by combining either the direct conjugate solution or the micelle solution with the LNPs and mixing at 60 °C for 1 h. Only the micelle mixing method produced stable particles with mRNA encapsulation above 80%. The LNPs were found to be stable up to 3 weeks (stored at 4 °C) as indicated by the RiboGreen assay and particle sizes. Targeted LNPs displayed relatively weak and neutral zeta potential. In vitro studies revealed enhanced cellular uptake of Green Fluorescent Protein (GFP) mRNA by targeted LNPs with similar transfection rates with thiol-maleimide and tetrazine-TCO chemistries showing around 97% GFP+ cells, compared to less than 30% in control groups at 6 h. Both targeted LNP formulations showed a significant rise in mean fluorescence intensity, achieving at least a 3-fold increase over the controls.

Hyperpolarized (HP) carbon-13 [13C] enables the specific investigation of dynamic metabolic and physiologic processes via in vivo MRI-based molecular imaging. As the leading HP metabolic agent, [1-13C]pyruvate plays a pivotal role due to its rapid tissue uptake and central role in cellular energetics. Dissolution dynamic nuclear polarization (d-DNP) is considered the gold standard method for the production of HP metabolic probes; however, development of a faster, less expensive technique could accelerate the translation of metabolic imaging via HP MRI to routine clinical use. Signal Amplification by Reversible Exchange in SHield Enabled Alignment Transfer (SABRE-SHEATH) achieves rapid hyperpolarization by using parahydrogen (p-H2) as the source of nuclear spin order. Currently, SABRE is clinically limited due to the toxicity of the iridium catalyst, which is crucial to the SABRE process. To mitigate Ir contamination, we introduce a novel iteration of the SABRE catalyst, incorporating bis(polyfluoroalkylated) imidazolium salts. This novel perfluorinated SABRE catalyst retained polarization properties while exhibiting an enhanced hydrophobicity. This modification allows the easy removal of the perfluorinated SABRE catalyst from HP [1-13C]-pyruvate after polarization in an aqueous solution, using the ReD-SABRE protocol. The residual Ir content after removal was measured via ICP-MS at 177 ppb, which is the lowest reported to date for pyruvate and is sufficiently safe for use in clinical investigations. Further improvement is anticipated once automated processes for delivery and recovery are initiated. SABRE-SHEATH using the perfluorinated SABRE catalyst can become an attractive low-cost alternative to d-DNP to prepare biocompatible HP [1-13C]-pyruvate formulations for in vivo applications in next-generation molecular imaging modalities.

The global rise in the incidence and severity of invasive fungal infections, particularly among immunocompromised and immunodeficient patients, has created an urgent need for rapid and accurate diagnostic techniques. Therefore, fungal-specific positron emission tomography imaging agents are increasingly in demand, as they offer the potential for early-stage detection of fungal infections. Recently, 2-deoxy-2-[18F]fluorocellobiose ([18F]FCB), a fluorine-18-labeled analog of cellobiose that is selectively metabolized by fungal pathogens possessing cellulose-degrading mechanisms (cellulolytic), was developed for the targeted imaging of Aspergillus infections. However, the final [18F]FCB contained less than 2% unreacted 2-deoxy-2-[18F]fluoroglucose ([18F]FDG), which can potentially interfere with image interpretation. Accordingly, this study aims to eliminate residual [18F]FDG from the final product by enzymatically converting it to [18F]FDG-6-phosphate through hexokinase-mediated phosphorylation. A Trasis AllInOne (Trasis AIO) module was used to automate the radiolabeling procedure. The reagent vials contain [18F]FDG, glucose-1-phosphate, cellobiose phosphorylase, adenosine triphosphate (ATP), and hexokinase. A Sep-Pak cartridge was used to purify the tracer. The overall radiochemical yield was 45–50% (n = 3, decay-corrected) in a 40 min synthesis time, with a radiochemical purity of >99% (no detectable [18F]FDG). This is a highly reliable protocol to produce current good manufacturing practice (cGMP)-compliant [18F]FCB for clinical PET imaging.

Spearman’s plot suggesting correlation between SUVmean (A) and SUVmax (B) intensity and GPC3 staining of HepG2, Hep3B, and Huh7 tumors (n = 2).

Abstract Background A major hurdle to the diagnosis of invasive fungal infections (IFIs), mainly caused by Aspergillus fumigatus (Af), remains the lack of timely and definite diagnosis. Several filamentous fungi express β-glucosidases (BGL) which convert cellobiose, a disaccharide, into 2 glucose units. Exploiting this pathway, we developed a novel tracer, 2-deoxy 2-[18F]fluorocellobiose (FCB) and showed it can specifically detect Af infections in vivo, with minimal background signal and radioactivity accumulation in live Af infection (Fig 1), but not in bacterial infection or sterile inflammation. In this study, we expanded the use of FCB to evaluate treatment response in Af infected mice. Specific detection of Af infection by FCB-PET imaging (A) Mechanism of action: FCB in the presence of β-glucosidase (BGL) producing fungi such as A. fumigatus (Af), is metabolized into FDG and glucose molecules. The resulting FDG is retained within the lesions containing metabolically active fungi. (B) Representative static PET/CT images at 120 mins after ∼7.4-9MBq bolus injection of FCB in mouse myositis models with live (right thigh) or heat-killed (HK) (left thigh) A. fumigatus (n=11) . The signal from the urinary bladder has been removed from the images for clarity purposes. SUVmean values show significantly higher PET signal in the site of live Af infection. ****, p < 0.0001 (unpaired t-test). GMS staining of tissues with live (hyphal structures) and HK (spores) fungi (Accepted for publication). Methods FCB was synthesized by enzymatic conversion of 2-deoxy-2-[18F]fluoroglucose (FDG). Mice were immunosuppressed using cyclophosphamide. Myositis infections were induced by intramuscular thigh injection of live Af conidia. Daily Voriconazole (VCZ) treatment was started on D1 for 3 weeks (40mg/kg, IP). Longitudinal FCB-PET was performed (D3, D14, D21) and standardized uptake values (SUVmean) were calculated. At the study endpoint, tissues were collected for Grocott’s Methenamine Silver (GMS) staining. Our results were compared to a similar previous treatment study where we used IV Amphotericin B (AmB) (15mg/kg). Evaluation of treatment response by FCB-PET imaging (A) Representative static PET/CT images of FCB uptake and SUV mean values in A. fumigatus myositis models (n=5) at different time-points after amphotericin B (AmB) treatment showing no appreciable decrease in PET signal over time. GMS staining of the thigh muscles at the terminal time-point show proliferating hyphae indicating sub-clinical efficacy of treatment. (B) Representative static PET/CT images of FCB uptake in A. fumigatus myositis models (n=12) at different time-points after voriconazole (VCZ) treatment showing decreased PET signal over time. SUVmean values show significant decrease in signal corresponding to the live infection focus (ANOVA mixed effects model) with significant treatment effect (p<0.0001). ** p<0.001; *p<0.05. GMS staining shows minimal hyphal growth indicating good control of infection. Results FCB-PET showed the initial spread and eventual decline in Af infection in VCZ treated mice with decreasing SUVmean values on D14 and D21 showing significant treatment effect (p< 0.0001, ANOVA mixed effects model) (Fig 2). As expected, VCZ treatment was more effective than AmB treatment which showed sub-clinical efficacy with minimal decrease in FCB-PET signal over time. GMS staining also showed decreased proliferating hyphae in the VCZ cohort unlike the AmB treated group. Conclusion We developed a novel Af-specific PET ligand, FCB, and demonstrated its ability to non-invasively diagnose Af infection in vivo and monitor treatment effect with different antifungals. Thus, FCB can also be used for repeated efficacy testing of novel therapeutics within the same cohort of animals. FCB is synthesized from commercial FDG and is a highly translatable Af-specific PET ligand. Further usefulness in other fungal infections is being evaluated. Disclosures All Authors: No reported disclosures

Leucine zipper-bearing kinase (LZK) is overexpressed in 20% of head and neck squamous cell carcinoma (HNSCC) cases and has emerged as a promising therapeutic target in this cancer subtype. LZK promotes HNSCC survival and proliferation by stabilizing c-MYC and GOF-p53 in kinase-dependent and -independent manners, respectively. Herein, we developed a new series of LZK degraders utilizing proteolysis-targeting chimera (PROTAC) technology by modulating the linker region or LZK warhead of LZK-targeting PROTAC-21A, previously developed by our laboratory. Among the 27 PROTACs synthesized and tested, PROTAC 17 was found to be the most potent, degrading LZK at 250 nM and suppressing HNSCC viability at 500 nM. In summary, our lead PROTAC effectively targeted LZK for proteasomal degradation and inhibited oncogenic activity in HNSCC cell lines with amplified LZK.

Fluorine-18 labeling of peptides and proteins is typically performed by an indirect labeling method. In this labeling approach, a labeled prosthetic group is prepared first and then conjugated to the proteins and peptides of interest. 6-[18F]fluoronicotinic acid-2,3,5,6-tetrafluorophenyl ester is a useful prosthetic group for indirect labeling. We have recently developed an efficient radiolabeling method, "fluorination on Sep-Pak," that enables the preparation of this prosthetic group with high radiochemical yield and purity in under 10 min. A variety of biomolecules have been radiolabeled using this prosthetic group. The radiolabeling procedure was either manual or semiautomated. However, a fully automated synthesis method is essential for successful clinical translation. Therefore, we developed a fully automated reproducible radiolabeling method to prepare fluorine-18-labeled albumin using the Trasis AllinOne module. The procedure was completed in 50 min. The overall radiochemical yield was 25%-36% (decay-corrected, n = 6) using 1 mg of albumin with a radiochemical purity > 98%.

The worldwide annual frequency and lethality of head and neck squamous cell carcinoma (HNSCC) is not improving, and thus, new therapeutic approaches are needed. Approximately 70% of HNSCC cases have either amplification or overexpression of MAP3K13, which encodes the kinase LZK. Here, we found that LZK is a therapeutic target in HNSCC and that small-molecule inhibition of its catalytic function decreased the viability of HNSCC cells with amplified MAP3K13. Inhibition of LZK suppressed tumor growth in MAP3K13-amplified xenografts derived from HNSCC patients. LZK stabilized the transcription factor c-MYC through its kinase activity and gain-of-function mutants of p53 in a kinase-independent manner. We designed a proteolysis-targeting chimera (PROTAC) that induced LZK degradation, leading to decreased abundance of both c-MYC and gain-of-function p53, and reduced the viability of HNSCC cells. Our findings demonstrate that LZK-targeted therapeutics, particularly PROTACs, may be effective in treating HNSCCs with MAP3K13 amplification.

PURPOSE:Hepatocellular carcinoma (HCC) is the third leading cause of cancer-related death and is characterized by poor survival rates and high recurrence after surgery. Glypican-3 (GPC3) is a proteoglycan highly expressed in HCC but absent in most normal tissue, making it an attractive diagnostic and therapeutic target. In this study, we introduce a second-generation GPC3-targeted single-domain antibody probe (ssHN3) bearing a positron-emitting isotope fluorine-18 (18F), ssHN3-Al[18F]F-RESCA (Al[18F]F-ssHN3), for HCC-selective PET imaging. In addition, we show its use as a functional imaging agent following focal tumor thermal ablation. EXPERIMENTAL DESIGN:Site-specific conjugation was used to synthesize the nanobody-based PET (immunoPET) probe ssHN3-Al[18F]F-RESCA. Binding affinity was determined using biolayer interferometry, and in vivo PET/CT imaging and biodistribution studies were performed in three liver cancer models with varying GPC3 expression (HepG2 > Hep3B > Huh7). Mice inoculated with HepG2 orthotopic liver tumors were also imaged before and 1 week after thermal tumor ablation to assess response. RESULTS:Our agent exhibited high purity (>98%), nanomolar affinity for GPC3, and tumor uptake corresponding to GPC3 expression on PET/CT and biodistribution studies. In orthotopic murine models of liver cancer, Al[18F]F-ssHN3 successfully distinguished between total versus subtotal thermal ablation, accurately identifying residual, viable disease. CONCLUSIONS:We successfully designed, engineered, and tested a GPC3-targeted 18F-labeled nanobody immunoPET agent, Al[18F]F-ssHN3, demonstrating that it can be used for same-day diagnostic imaging in murine models of liver cancer. Importantly, this agent could address the limitations of current imaging methods by detecting residual disease after thermal ablation and other locoregional therapies.

Biodistribution of Al[18F]F-ssHN3 in mice bearing HepG2-GPC3-/- xenografts at 1, 2, and 3 h after administration (n = 4-6 / time point).

Crosslinking mass spectrometry (MS) is a powerful approach for probing protein structures. However, most widely used crosslinkers rely on N-hydroxysuccinimide (NHS) esters, restricting reactivity primarily to lysine residues and protein N-termini, and rendering them incompatible with many amine-containing buffers (e.g., Tris) and key biochemical cofactors (e.g., ATP). To address these limitations, we introduce two novel vinyl-sulfone-based crosslinkers. Alkyne-BVSC is an enrichable, homobifunctional crosslinker featuring an acid-cleavable alkyne handle for downstream peptide enrichment. VSD is a heterobifunctional crosslinker combining a vinyl sulfone with a diazirine moiety for UV-activated photo-crosslinking. Both reagents are synthetically accessible from inexpensive precursors and retain reactivity in amine-rich biochemical environments. We show that vinyl sulfones react with cysteine, histidine, and lysine residues, thereby expanding crosslinkable residues beyond those accessible to NHS-esters. Moreover, we develop a stub-based post-search filtering strategy that leverages the MS-cleavable nature of vinyl sulfone linkages to improve crosslink identification sensitivity. Together, these advances establish vinyl-sulfone-based crosslinkers as versatile and complementary tools for structural proteomics.

B7-H3, an immunomodulatory protein overexpressed in many cancers, is associated with tumor aggressiveness and poor prognosis, making it a crucial target for imaging to elucidate its role in cancer progression and guide therapeutic interventions. This study employed PET imaging to investigate the in vivo delivery and pharmacokinetics of two anti-B7-H3 antibodies, Ab-1 and Ab-2, in mouse xenograft models with varying B7-H3 expression levels. The antibodies were radiolabeled with [89Zr]Zr and evaluated through PET imaging, biodistribution studies, and in vitro assays to assess binding, tumor uptake, and retention. [89Zr]Zr-Ab-1 demonstrated high initial tumor uptake in B7-H3 positive xenografts but exhibited unexpected decreasing retention over time. This clearance was likely attributed to proteolytic cleavage mediated by matrix metalloproteinases in the tumor and the tumor microenvironment. Conversely, [89Zr]Zr-Ab-2 showed more stable tumor retention but lower overall uptake. Further investigation revealed that Ab-1 had affinity for both 4Ig and 2Ig B7-H3 isoforms, while Ab-2 bound exclusively to the 4Ig isoform. This differential binding to B7-H3 isoforms may explain the observed variations in tumor uptake and retention between the two antibodies. The study provides insights into the complex dynamics of B7-H3 targeted antibodies in vivo, highlighting how antibody characteristics, including isoform-specific binding, and tumor factors influence their behavior. These findings have potential implications for optimizing radiotherapy strategies, suggesting the possibility of tailored approaches based on antibody properties and tumor biology.

Biodistribution of Al[18F]F-ssHN3 in mice bearing HepG2 xenografts at 1, 2, and 3 h after administration (n = 4-5 / time point).

Gastro-enteropancreatic neuroendocrine neoplasms (GEP-NENs) are a diverse family of tumors which can originate throughout the gastrointestinal tract. Despite a designation as a rare cancer, the incidence of GEP-NENs has increased over the last few decades. There has been difficulty in developing new therapies due to a lack of targetable mutations present in these tumors. One potential target is somatostatin receptor 2 (SSTR2), which is expressed in many of these cancers. Somatostatin analogues and peptide-targeted radiotherapies have been developed to target these tumors, but treatment acquired resistance is common and SSTR2 expression is often lost during progression from less aggressive, lower grade tumors towards more aggressive, higher-grade tumors. Due to this, additional therapies are needed to target these tumors to improve patient outcomes. To address this gap, we conducted a high-throughput screen of high grade pancreatic NEN cell lines BON-1 and QGP-1 to identify potentially therapeutic small molecules using the MIPE 5.0 library at the National Center for Advancing Translational Sciences (NCATS). The top 20 molecules from classes including proteosome inhibitors, BRAF/MEK inhibitors, NAMPT inhibitors, and topoisomerase inhibitors were further selected for repeat screening with the addition of two well-differentiated cell lines NT-18P and NT-3 and GEP-NEN patient tumor organoids (PTO, n=51 tumors). Results from these screens, comparison of targets to known mutation profiles of high-throughput models, and analysis of current conjugation methods were used to select exatecan, a topoisomerase 1 inhibitor, for usage as a payload for a conjugated therapy. Exatecan showed high statistically significant cytotoxicity with PTO grade (grade 1 IC50 average=331 nM, 206 nM for grade 2, 90 nM for grade 3, p=0.01), but not with origin (pancreatic NEN IC50=157 nM vs small bowel NEN=356 nM, p=0.10). Exatecan and its derivative deruxatecan has already been incorporated into the treatment paradigm for HER2+ breast cancer. In partnership with the Chemical Synthesis Center at the National Institute of Health, click chemistry to incorporate cleavable crosslinkers was used to create three SSTR2-targeted drug conjugates based on octreatate. Finally, each of the compounds were shown to effectively target cell lines with SSTR2 expression, while cell lines with no SSTR2 expression demonstrated significantly lower sensitivity towards the compounds. In conclusion, we have created a series of compounds which demonstrate optimal chemical characteristics and promising targeting of SSTR2+ GEP-NENs. Next steps include the analysis of binding affinity for SSTR2, as well as stability testing for usage as a therapy and the incorporation of SSTR2+ xenograft models for treatment efficacy. Steven Donald Forsythe, Srujana V. Yellapragada, Tracey Pu, Ken C. Cheng, Freddy E. Escorcia, Rolf E. Swenson, James P. Madigan, Samira M. Sadowski. Development of SSTR2-peptide conjugated exatecan for therapy of advanced GEP-NENs [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 4293.

Tumor-to-tissue ratios of Al[18F]F-ssHN3 in orthotopic HepG2 liver cancer model calculated from ex vivo biodistribution (n = 4 / time point).