BxPC-3 Xenografts Research Articles

Investigation of Copper-64-Based Host-Guest Chemistry Pretargeted Positron Emission Tomography.

Pretargeting is a technique that uses macromolecules as targeting agents for nuclear imaging and therapy with the goal of reducing the radiation toxicity to healthy tissues often associated with directly radiolabeled macromolecules. In pretargeting, a macromolecule is radiolabeled in vivo at the target site using a radiolabeled small molecule (radioligand) that interacts with the macromolecule with high specificity. We report an investigation of host-guest chemistry-driven pretargeting using copper-64 radiolabeled ferrocene (Fc; guest) compounds and a cucurbit[7]uril (CB7; host) molecule functionalized carcinoembryonic antigen targeting hT84.66-M5A monoclonal antibody (CB7-M5A). Two novel ferrocene-based radioligands ([64Cu]Cu-NOTA-PEG3-Fc and [64Cu]Cu-NOTA-PEG7-Fc) were prepared, and their in vitro stability, pharmacokinetic in vivo profile in healthy mice, and pretargeting performance in a subcutaneous BxPC3 human pancreatic cancer cell xenograft mouse model were compared. The antibody dosing was optimized using a zirconium-89 radiolabeled M5A antibody ([89Zr]Zr-DFO-M5A) in a BxPC3 xenograft model, and the dosimetry of [89Zr]Zr-DFO-M5A and the pretargeting approach were compared. Finally, the effects of varying lag times up to 9 days between CB7-M5A and radioligand injection were investigated. In vivo pretargeting studies with both ferrocene radioligands resulted in specific tumor uptake (p = 0.0006 and p = 0.003) and also showed that the host-guest-based pretargeting approach excels with extended lag times up to 9 days with good tumor localization, suggesting that host-guest pretargeting may be suitable for use without clearing agents which have complicated clinical application of this technique. To our knowledge, the reported lag time of 9 days is the longest investigated lag time in any reported pretargeting studies.

Targeting Tumor Cells Overexpressing the Human Epidermal Growth Factor Receptor 3 with Potent Drug Conjugates Based on Affibody Molecules.

Increasing evidence suggests that therapy targeting the human epidermal growth factor receptor 3 (HER3) could be a viable route for targeted cancer therapy. Here, we studied a novel drug conjugate, ZHER3-ABD-mcDM1, consisting of a HER3-targeting affibody molecule, coupled to the cytotoxic tubulin polymerization inhibitor DM1, and an albumin-binding domain for in vivo half-life extension. ZHER3-ABD-mcDM1 showed a strong affinity to the extracellular domain of HER3 (KD 6 nM), and an even stronger affinity (KD 0.2 nM) to the HER3-overexpressing pancreatic carcinoma cell line, BxPC-3. The drug conjugate showed a potent cytotoxic effect on BxPC-3 cells with an IC50 value of 7 nM. Evaluation of a radiolabeled version, [99mTc]Tc-ZHER3-ABD-mcDM1, showed a relatively high rate of internalization, with a 27% internalized fraction after 8 h. Further in vivo evaluation showed that it could target BxPC-3 (pancreatic carcinoma) and DU145 (prostate carcinoma) xenografts in mice, with an uptake peaking at 6.3 ± 0.4% IA/g at 6 h post-injection for the BxPC-3 xenografts. The general biodistribution showed uptake in the liver, lung, salivary gland, stomach, and small intestine, organs known to express murine ErbB3 naturally. The results from the study show that ZHER3-ABD-mcDM1 is a highly potent and selective drug conjugate with the ability to specifically target HER3 overexpressing cells. Further pre-clinical and clinical development is discussed.

Timosaponin A3 Inhibits Palmitate and Stearate through Suppression of SREBP-1 in Pancreatic Cancer

Timosaponin A3 (TA3) was demonstrated as a potent anticancer chemical by several studies. Although the effects of inhibiting growth, metastasis, and angiogenesis in various cancer cells were demonstrated through multiple mechanisms, the pharmacological mechanism of TA3 shown in pancreatic cancer (PC) is insufficient compared to other cancers. In this study, we aimed to explore the key molecular mechanisms underlying the growth inhibitory effects of TA3 using PC cells and a xenograft model. First, from the microarray results, we found that TA3 regulated INSIG-1 and HMGCR in BxPC-3 cells. Furthermore, we showed that inhibition of sterol regulatory element-binding protein-1 (SREBP-1) by TA3 reduced the fatty acid synthases FASN and ACC, thereby controlling the growth of BxPC-3 cells. We also tried to find mechanisms involved with SREBP-1, such as Akt, Gsk3β, mTOR, and AMPK, but these were not related to SREBP-1 inhibition by TA3. In the BxPC-3 xenograft model, the TA3 group had more reduced tumor formation and lower toxicity than the gemcitabine group. Interestingly, the level of the fatty acid metabolites palmitate and stearate were significantly reduced in the tumor tissue in the TA3 group. Overall, our study demonstrated that SREBP-1 was a key transcription factor involved in pancreatic cancer growth and it remained a precursor form due to TA3, reducing the adipogenesis and growth in BxPC-3 cells. Our results improve our understanding of novel mechanisms of TA3 for the regulation of lipogenesis and provide a new approach to the prevention and treatment of PC.

A Comparison of Evans Blue and 4-(p-Iodophenyl)butyryl Albumin Binding Moieties on an Integrin αvβ6 Binding Peptide.

Serum albumin binding moieties (ABMs) such as the Evans blue (EB) dye fragment and the 4-(p-iodophenyl)butyryl (IP) have been used to improve the pharmacokinetic profile of many radiopharmaceuticals. The goal of this work was to directly compare these two ABMs when conjugated to an integrin αvβ6 binding peptide (αvβ6-BP); a peptide that is currently being used for positron emission tomography (PET) imaging in patients with metastatic cancer. The ABM-modified αvβ6-BP peptides were synthesized with a 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetracetic acid (DOTA) chelator for radiolabeling with copper-64 to yield [64Cu]Cu DOTA-EB-αvβ6-BP ([64Cu]1) and [64Cu]Cu DOTA-IP-αvβ6-BP ([64Cu]2). Both peptides were evaluated in vitro for serum albumin binding, serum stability, and cell binding and internalization in the paired engineered melanoma cells DX3puroβ6 (αvβ6 +) and DX3puro (αvβ6 −), and pancreatic BxPC-3 (αvβ6 +) cells and in vivo in a BxPC-3 xenograft mouse model. Serum albumin binding for [64Cu]1 and [64Cu]2 was 53–63% and 42–44%, respectively, with good human serum stability (24 h: [64Cu]1 76%, [64Cu]2 90%). Selective αvβ6 cell binding was observed for both [64Cu]1 and [64Cu]2 (αvβ6 (+) cells: 30.3–55.8% and 48.5–60.2%, respectively, vs. αvβ6 (−) cells <3.1% for both). In vivo BxPC-3 tumor uptake for both peptides at 4 h was 5.29 ± 0.59 and 7.60 ± 0.43% ID/g ([64Cu]1 and [64Cu]2, respectively), and remained at 3.32 ± 0.46 and 4.91 ± 1.19% ID/g, respectively, at 72 h, representing a >3-fold improvement over the non-ABM parent peptide and thereby providing improved PET images. Comparing [64Cu]1 and [64Cu]2, the IP-ABM-αvβ6-BP [64Cu]2 displayed higher serum stability, higher tumor accumulation, and lower kidney and liver accumulation, resulting in better tumor-to-organ ratios for high contrast visualization of the αvβ6 (+) tumor by PET imaging.

O-P03 A composite polymeric nanoparticle as a sensitiser for sonodynamic therapy (SDT)-based treatment of pancreatic cancer

Abstract Background Pancreatic cancer remains one of the most recalcitrant forms of cancer with poor prognosis and limited treatment options. SDT is a novel, targeted approach to the treatment of solid tumours. Based on the generation of cytotoxic reactive oxygen species (ROS) following the exposure of a sonosensitiser to ultrasound, the approach is designed to extracorporeally target less accessible lesions. Here we describe the production of a poly(lactic-co-glycolic acid) (PLGA), polyethyleneimine (PEI), Rose Bengal (RB) and indocyanine green (ICG) containing composite nanoparticles and describe their use in SDT-mediated treatment of pancreatic cancer using both in vitro and in vivo target models. Methods Nanoparticles were prepared using an oil in water emulsion and solvent diffusion-based approach. These were designated RB-ICGNP. In vitro SDT treatment consisted of exposing BxPC3 (human PDAC cells), T110029 (murine PDAC cells) or hPSC (immortalised human pancreatic stellate cells) to RB-ICGNP and subsequently treating with ultrasound for 30 s at a frequency of 1 MHz, a power density of 3.0 W/cm2 (SATP) using a duty cycle of 50% at a pulse repetition frequency of 100 Hz. For in vivo studies, BxPC3 (xenograft) and T110029 (syngeneic) tumours were treated with a power density of 3.5 W/cm2 ultrasound for 3.5 min. Results Conclusions Using in vitro and in vivo (human xenograft and murine syngeneic) models of pancreatic cancer, RB-ICGNP composite nanoparticles may be employed as a sensitiser for SDT-based treatment of pancreatic cancer. Since pancreatic stellate cells were more sensitive to SDT, the latter may have an impact on tumour stroma. Staining of residual tumour tissues from SDT-treated animals for connective tissue (stroma) confirmed the latter. Since tumour stroma presents a significant challenge to treatment of pancreatic cancer and represents a negative prognostic marker, the impact delivered by SDT may be exploited to potentiate alternative therapeutic approaches.

Evaluation of Copper-64-Labeled αvβ6-Targeting Peptides: Addition of an Albumin Binding Moiety to Improve Pharmacokinetics.

The incorporation of non-covalent albumin binding moieties (ABMs) into radiotracers results in increased circulation time, leading to a higher uptake in the target tissues such as the tumor, and, in some cases, reduced kidney retention. We previously developed [18F]AlF NOTA-K(ABM)-αvβ6-BP, where αvβ6-BP is a peptide with high affinity for the cell surface receptor integrin αvβ6 that is overexpressed in several cancers, and the ABM is an iodophenyl-based moiety. [18F]AlF NOTA-K(ABM)-αvβ6-BP demonstrated prolonged blood circulation compared to the non-ABM parent peptide, resulting in high, αvβ6-targeted uptake with continuously improving detection of αvβ6(+) tumors using PET/CT. To further extend the imaging window beyond that of fluorine-18 (t1/2 = 110 min) and to investigate the pharmacokinetics at later time points, we radiolabeled the αvβ6-BP with copper-64 (t1/2 = 12.7 h). Two peptides were synthesized without (1) and with (2) the ABM and radiolabeled with copper-64 to yield [64Cu]1 and [64Cu]2, respectively. The affinity of [natCu]1 and [natCu]2 for the integrin αvβ6 was assessed by enzyme-linked immunosorbent assay. [64Cu]1 and [64Cu]2 were evaluated in vitro (cell binding and internalization) using DX3puroβ6 (αvβ6(+)), DX3puro (αvβ6(-)), and pancreatic BxPC-3 (αvβ6(+)) cells, in an albumin binding assay, and for stability in both mouse and human serum. In vivo (PET/CT imaging) and biodistribution studies were done in mouse models bearing either the paired DX3puroβ6/DX3puro or BxPC-3 xenograft tumors. [64Cu]1 and [64Cu]2 were synthesized in ≥97% radiochemical purity. In vitro, [natCu]1 and [natCu]2 maintained low nanomolar affinity for integrin αvβ6 (IC50 = 28 ± 3 and 19 ± 5 nM, respectively); [64Cu]1 and [64Cu]2 showed comparable binding to αvβ6(+) cells (DX3puroβ6: ≥70%, ≥42% internalized; BxPC-3: ≥19%, ≥12% internalized) and ≤3% to the αvβ6(-) DX3puro cells. Both radiotracers were ≥98% stable in human serum at 24 h, and [64Cu]2 showed a 6-fold higher binding to human serum protein than [64Cu]1. In vivo, selective uptake in the αvβ6(+) tumors was observed with tumor visualization up to 72 h for [64Cu]2. A 3-5-fold higher αvβ6(+) tumor uptake of [64Cu]2 vs [64Cu]1 was observed throughout, at least 2.7-fold improved BxPC-3-to-kidney and BxPC-3-to-blood ratios, and 2-fold improved BxPC-3-to-stomach ratios were noted for [64Cu]2 at 48 h. Incorporation of an iodophenyl-based ABM into the αvβ6-BP ([64Cu]2) prolonged circulation time and resulted in improved pharmacokinetics, including increased uptake in αvβ6(+) tumors that enabled visualization of αvβ6(+) tumors up to 72 h by PET/CT imaging.

Combination of ONC201 and TLY012 induces selective, synergistic apoptosis in vitro and significantly delays PDAC xenograft growth in vivo

ABSTRACT The five-year survival rate for pancreatic ductal adenocarcinoma (PDAC) has remained a dismal 9% for approximately 40 years with an urgent need for novel therapeutic interventions. ONC201 is the founding member of the imipridone class, comprised of orally bioavailable small molecules that have shown efficacy in multiple tumor types both in animal models and in Phase I/II clinical trials. ONC201 is a potent inducer of the tumor necrosis factor related apoptosis inducing ligand (TRAIL) pathway. TRAIL is an innate immune mechanism which induces programmed cell death of cancer cells. We observed that PDAC cells upregulated ATF4, CHOP, and DR5 after treatment with ONC201. This occurred in cell lines that are susceptible to ONC201-induced apoptosis and in ones that are not. In response to ONC201, PDAC cells downregulated anti-apoptotic proteins including c-FLIP, BclXL, XIAP, cIAP1, and survivin. We hypothesized that TRAIL receptor agonists might induce selective, synergistic apoptosis in pancreatic cancer cell lines treated with ONC201. We screened 7 pancreatic cancer cell lines and found synergy with ONC201 and rhTRAIL or the novel TRAIL receptor agonist TLY012 in 6 of the 7 cell lines tested. In vivo experiments using BxPC3 and HPAFII xenograft models showed that the combination of ONC201 plus TLY012 significantly delays tumor growth as compared to controls. Immunohistochemical analysis of the tumors after three doses of the combination showed significantly increased cleavage of caspase 3 in vivo as compared to controls. Taken together, the preclinical efficacy of ONC201 and TLY012 represents a novel therapeutic option for further testing in pancreatic cancer patients. This combination showed marked efficacy in tumor cells that are both sensitive and resistant to the pro-apoptotic effects of ONC201, providing rationale to further investigate the combination of ONC201 plus TLY012 in patients with pancreatic cancer.

HER3 PET Imaging: 68Ga-Labeled Affibody Molecules Provide Superior HER3 Contrast to 89Zr-Labeled Antibody and Antibody-Fragment-Based Tracers.

Simple SummaryHER3 is a known driver for oncogenesis and therapy resistance in solid cancers. PET imaging could be a useful tool to non-invasively detect and monitor HER3 expression and aid in the selection of patients for HER3-targeted therapy. PET tracers based on therapeutic antibodies have thus far shown limited success in reliably imaging HER3-expressing tumors in clinical trials. Smaller-sized tracers specifically designed for imaging might be needed for higher contrast imaging and sufficient sensitivity. Our group has previously studied the use of radiolabeled affibody molecules for imaging of HER3 expression. In the present study, we compared four different types of potential PET tracers for imaging of HER3 expression in a preclinical model. We demonstrated that the affibody-based tracer, [68Ga]Ga-ZHER3, could provide overall superior imaging contrast to antibody- and antibody-fragment-based tracers shortly after injection. Our results indicate that HER3-targeting affibody molecules are promising agents for PET imaging of HER3 expression.HER3 (human epidermal growth factor receptor type 3) is a challenging target for diagnostic radionuclide molecular imaging due to the relatively modest overexpression in tumors and substantial expression in healthy organs. In this study, we compared four HER3-targeting PET tracers based on different types of targeting molecules in a preclinical model: the 89Zr-labeled therapeutic antibody seribantumab, a seribantumab-derived F(ab)2-fragment labeled with 89Zr and 68Ga, and the 68Ga-labeled affibody molecule [68Ga]Ga-ZHER3. The novel conjugates were radiolabeled and characterized in vitro using HER3-expressing BxPC-3 and DU145 human cancer cells. Biodistribution was studied using Balb/c nu/nu mice bearing BxPC-3 xenografts. HER3-negative RAMOS xenografts were used to demonstrate binding specificity in vivo. Autoradiography was conducted on the excised tumors. nanoPET/CT imaging was performed. New conjugates specifically bound to HER3 in vitro and in vivo. [68Ga]Ga-DFO-seribantumab-F(ab’)2 was considered unsuitable for imaging due to the low stability and high uptake in normal organs. The highest tumor-to-non-tumor contrast with [89Zr]Zr-DFO-seribantumab and [89Zr]Zr-DFO-seribantumab-F(ab’)2 was achieved at 96 h and 48 h pi, respectively. Despite lower tumor uptake, [68Ga]Ga-ZHER3 provided the best imaging contrast due to the fastest clearance from blood and normal organs. The results of our study suggest that affibody-based tracers are more suitable for PET imaging of HER3 expression than antibody- and antibody-fragment-based tracers.

The next-generation pan-RAF inhibitor, KIN-2787, is active in class II and class III BRAF mutant models.

3116 Background: Oncogenic BRAF gene alterations, leading to aberrantly activated BRAF monomers (Class I mutations) or dimers (Class II and Class III mutations), are observed in approximately 6% of all human cancers. First-generation BRAF inhibitors targeting Class I BRAF mutants, including dabrafenib, encorafenib, and vemurafenib, provide significant clinical benefit to patients with BRAF V600 mutation-driven melanoma and select solid tumors as monotherapies or in combination with other targeted therapies. The currently approved BRAF inhibitors have not, however, proven to be effective in patients with Class II or III BRAF alterations which account for a large proportion (34%) of BRAF mutations. KIN-2787 is an orally available, potent and selective small molecule pan-RAF inhibitor specifically designed to inhibit Class II and III BRAF dimers, in addition to Class I mutants. Methods: The efficacy and tolerability of the pan-RAF inhibitor, KIN-2787, was evaluated in vitro and in vivo in Class I, II, and III BRAF mutation-driven human cancer models. Results: In biochemical assays, KIN-2787 showed low nanomolar to picomolar potency against RAF1, BRAF, and ARAF (IC50 0.06-3.46 nM) with minimal activity towards non-RAF kinases. In cell-based assays, KIN-2787 inhibited RAF activity, as measured by inhibition of downstream ERK phosphorylation (pERK), across multiple BRAF mutant cancer cell lines. Class II and III BRAF mutant cell lines were the most responsive when treated with KIN-2787 (IC50 &lt; 50 nM); 19- and 7-fold more sensitive compared to cells harboring wild-type BRAF, respectively. Dose-dependent inhibition of A-375 (Class I), BxPC-3 (Class II), and WM3629 (Class III) BRAF mutant human xenograft tumor growth was attained with daily KIN-2787 treatment and was well-tolerated. A trend towards greater tumor responses was observed with twice daily (BID) compared to once daily (QD) dosing of KIN-2787; however, the two dosing regimens led to similar tumor growth inhibition (TGI) and regressions (mean TGI up to 101-118%; p ≤0.0001) at equivalent total daily doses. Furthermore, KIN-2787 led to a significant in vivo pharmacodynamic response using either regimen, however, prolonged target coverage, as measured by pERK, was achieved with BID dosing. The impact of KIN-2787 treatment on additional biomarkers, including transcriptional changes and MAPK pathway modulation in cell-based models and patient-derived samples, will be presented at the meeting. Conclusions: KIN-2787 is a next-generation pan-RAF inhibitor with pronounced in vitro and in vivo activity against human cancers driven by Class II and III BRAF mutations. A phase 1 dose escalation and expansion clinical trial evaluating the safety and efficacy of KIN-2787 monotherapy in patients with advanced or metastatic solid tumors harboring BRAF gene alterations, including Class II and III mutations, is expected to initiate in 2021.

86/90Y-Labeled Monoclonal Antibody Targeting Tissue Factor for Pancreatic Cancer Theranostics.

Pancreatic cancer is highly aggressive, with a median survival time of less than 6 months and a 5-year overall survival rate of around 7%. The poor prognosis of PaCa is largely due to its advanced stage at diagnosis and the lack of efficient therapeutic options. Thus, the development of an efficient, multifunctional PaCa theranostic system is urgently needed. Overexpression of tissue factor (TF) has been associated with increased tumor growth, angiogenesis, and metastasis in many malignancies, including pancreatic cancer. Herein, we propose the use of a TF-targeted monoclonal antibody (ALT836) conjugated with the pair 86/90Y as a theranostic agent against pancreatic cancer. For methods, serial PET imaging with 86Y-DTPA-ALT836 was conducted to map the biodistribution the tracer in BXPC-3 tumor-bearing mice. 90Y-DTPA-ALT836 was employed as a therapeutic agent that also allowed tumor burden monitoring through Cherenkov luminescence imaging. The results were that the uptake of 86Y-DTPA-ALT836 in BXPC-3 xenograft tumors was high and increased over time up to 48 h postinjection (p.i.), corroborated through ex vivo biodistribution studies and further confirmed by Cherenkov luminescence Imaging. In therapeutic studies, 90Y-DTPA-ALT836 was found to slow tumor growth relative to the control groups and had significantly smaller (p < 0.05) tumor volumes 1 day p.i. Histological analysis of ex vivo tissues revealed significant damage to the treated tumors. The conclusion is that the use of the 86/90Y theranostic pair allows PET imaging with excellent tumor-to-background contrast and treatment of TF-expressing pancreatic tumors with promising therapeutic outcomes.

Benefit of Later-Time-Point PET Imaging of HER3 Expression Using Optimized Radiocobalt-Labeled Affibody Molecules.

HER3-binding affibody molecules are a promising format for visualization of HER3 expression. Cobalt-55, a positron-emitting isotope, with a half-life of 17.5 h, allows for next-day imaging. We investigated the influence of the charge of the radiocobalt–chelator complex on the biodistribution of anti-HER3 affibody molecule (HE)3-ZHER3 and compared the best radiocobalt-labeled variant with a recently optimized gallium-labeled variant. Affibody conjugates (HE)3-ZHER3-X (X = NOTA, NODAGA, DOTA, DOTAGA) were labeled with [57Co]Co (surrogate for 55Co). Affinity measurements, binding specificity and cellular processing were studied in two HER3-expressing cancer cell lines. Biodistribution was studied 3 and 24 h post-injection (pi) in mice with HER3-expressing BxPC-3 xenografts and compared to [68Ga]Ga-(HE)3-ZHER3-NODAGA. Micro-single-photon emission tomography/computed tomography (microSPECT/CT) and micro-positron emission tomography/computed tomography (microPET/CT) imaging was performed 3 and 24 h pi. Stably labeled conjugates bound to HER3 with subnanomolar affinity. [57Co]Co-(HE)3-ZHER3-DOTA had the best tumor retention and a significantly lower concentration in blood than other conjugates, leading to superior tumor-to-blood and tumor-to-liver ratios 24 h pi. Compared to [68Ga]Ga-(HE)3-ZHER3-NODAGA 3 h pi, [57Co]Co-(HE)3-ZHER3-DOTA provided superior imaging contrast in liver 24 h pi. Concluding, the composition and charge of the [57Co]Co–chelator complex influenced the uptake in tumors and normal tissue. [57Co]Co-(HE)3-ZHER3-DOTA provided the best imaging properties among the cobalt-labeled conjugates. Delayed imaging of HER3 expression with [57Co]Co-(HE)3-ZHER3-DOTA improved imaging contrast compared to early-time-point imaging with [68Ga]Ga-(HE)3-ZHER3-NODAGA.

Detecting TRA-1-60 in Cancer via a Novel Zr-89 Labeled ImmunoPET Imaging Agent.

TRA-1-60 (TRA) is a cell-surface antigen implicated in drug resistance, relapse, and recurrence. Its expression has been reported in breast, prostate, pancreatic, ovarian tumors, and follicular lymphoma, which paved the development of the therapeutic antibody, Bstrongomab (Bsg), and its drug conjugates. Because patient selection is critical to achieve clinical benefit, a noninvasive imaging agent to select TRA+ lesions in patients is needed. Herein, we report the development of the immunopositron emission tomography (immunoPET) radiotracer 89Zr-radiolabeled Bsg and its potential to delineate TRA+ tumors. Bsg was conjugated to the bifunctional chelator desferrioxamine (DFO) and radiolabeled with [89Zr]Zr-oxalate. [89Zr]Zr-DFO-Bsg was characterized in vitro and evaluated in vivo for uptake and specificity in high and low TRA-expressing BxPC-3 pancreatic and PC-3 prostate cancer models, respectively. Uptake was compared against [89Zr]Zr-DFO-IgG, a nonspecific control radiotracer. Immunohistochemical (IHC) staining of patient cancer tissues using Bsg was performed to explore its clinical significance. A specific activity of 0.18 ± 0.01 GBq/mg (4.8 ± 0.3 mCi/mg) was obtained for [89Zr]Zr-DFO-Bsg. BxPC-3 xenografts exhibited three-fold higher radiotracer uptake compared to [89Zr]Zr-DFO-IgG. Competitive saturation studies using BxPC-3 xenografts further confirmed tracer specificity. The TRA-specific probe had lower accumulation in PC-3 xenografts. Ex vivo autoradiographs correlated with TRA expression from the histopathology of the resected tumor xenografts. Additionally, patient cancer tissues demonstrated positive staining with Bsg with metastatic lesions exhibiting the highest staining. This study demonstrates the potential of [89Zr]Zr-DFO-Bsg as an imaging agent for noninvasive detection of TRA+ tumors.

Influence of Residualizing Properties of the Radiolabel on Radionuclide Molecular Imaging of HER3 Using Affibody Molecules.

Human epidermal growth factor receptor type 3 (HER3) is an emerging therapeutic target in several malignancies. To select potential responders to HER3-targeted therapy, radionuclide molecular imaging of HER3 expression using affibody molecules could be performed. Due to physiological expression of HER3 in normal organs, high imaging contrast remains challenging. Due to slow internalization of affibody molecules by cancer cells, we hypothesized that labeling (HE)3-ZHER3:08698-DOTAGA affibody molecule with non-residualizing [125I]-N-succinimidyl-4-iodobenzoate (PIB) label would improve the tumor-to-normal organs ratios compared to previously reported residualizing radiometal labels. The [125I]I-PIB-(HE)3-ZHER3:08698-DOTAGA was compared side-by-side with [111In]In-(HE)3-ZHER3:08698-DOTAGA. Both conjugates demonstrated specific high-affinity binding to HER3-expressing BxPC-3 and DU145 cancer cells. Biodistribution in mice bearing BxPC-3 xenografts at 4 and 24 h pi showed faster clearance of the [125I]I-PIB label compared to the indium-111 label from most tissues, except blood. This resulted in higher tumor-to-organ ratios in HER3-expressing organs for [125I]I-PIB-(HE)3-ZHER3:08698-DOTAGA at 4 h, providing the tumor-to-liver ratio of 2.4 ± 0.3. The tumor uptake of both conjugates was specific, however, it was lower for the [125I]I-PIB label. In conclusion, the use of non-residualizing [125I]I-PIB label for HER3-targeting affibody molecule provided higher tumor-to-liver ratio than the indium-111 label, however, further improvement in tumor uptake and retention is needed.

Increase in negative charge of 68Ga/chelator complex reduces unspecific hepatic uptake but does not improve imaging properties of HER3-targeting affibody molecules

Upregulation of the human epidermal growth factor receptor type 3 (HER3) is a common mechanism to bypass HER-targeted cancer therapy. Affibody-based molecular imaging has the potential for detecting and monitoring HER3 expression during treatment. In this study, we compared the imaging properties of newly generated 68Ga-labeled anti-HER3 affibody molecules (HE)3-ZHER3-DOTA and (HE)3-ZHER3-DOTAGA with previously reported [68Ga]Ga-(HE)3-ZHER3-NODAGA. We hypothesized that increasing the negative charge of the gallium-68/chelator complex would reduce hepatic uptake, which could lead to improved contrast of anti-HER3 affibody-based PET-imaging of HER3 expression. (HE)3-ZHER3-X (X = DOTA, DOTAGA) were produced and labeled with gallium-68. Binding of the new conjugates was specific in HER3 expressing BxPC-3 and DU145 human cancer cells. Biodistribution and in vivo specificity was studied in BxPC-3 xenograft bearing Balb/c nu/nu mice 3 h pi. DOTA- and DOTAGA-containing conjugates had significantly higher concentration in blood than [68Ga]Ga-(HE)3-ZHER3-NODAGA. Presence of the negatively charged 68Ga-DOTAGA complex reduced the unspecific hepatic uptake, but did not improve overall biodistribution of the conjugate. [68Ga]Ga-(HE)3-ZHER3-DOTAGA and [68Ga]Ga-(HE)3-ZHER3-NODAGA had similar tumor-to-liver ratios, but [68Ga]Ga-(HE)3-ZHER3-NODAGA had the highest tumor uptake and tumor-to-blood ratio among the tested conjugates. In conclusion, [68Ga]Ga-(HE)3-ZHER3-NODAGA remains the favorable variant for PET imaging of HER3 expression.

76P - Proton-sensitizing effect of small molecule inhibitor of P300 histone acetyltransferase C646 in human pancreatic cancer cells

BackgroundHistone acetylation plays an important role in regulating the chromatin structure and is tightly regulated by histone acetyltransferases (HAT) and histone deacetylases (HDAC). Recent studies have demonstrated that several chromatin-regulating proteins can modulate cellular responses to other cytotoxic modalities including ionizing radiation and chemotherapeutic drugs. Even though histone acetylation is one of the key mechanisms of epigenetic regulation, relatively little is known about the cancer therapeutic potential of HAT inhibitors. This is in stark contrast to the well-studied effects of HDAC inhibitors. In this study, we investigated the proton beam-sensitizing effect of C646, a selective small molecule inhibitor of p300 histone acetyltransferase in human pancreatic cancer cells. MethodsCell viability assay (CCK-8; Dojindo), Clonogenic survival assay, Cell cycle analysis (PI staining). Annexin V-FITC and PI staining, γ-H2AX foci, Western blot, BxPC-3 Xenograft tumor model, TUNEL assay. ResultsAsPC-1, BxPC-3 and Mia-paca2 cells exhibited increase in radiosensitivity when exposed to C646 at all doses of proton beam tested. C646 pre-treatment induce led to increase of sub-G1 population and abolishment of G2/M arrest. Flow cytometry analysis with annexin V staining and Western blot analysis showed that The pre-treatment with C646 significantly enhanced proton-induced apoptotic cell death through the down-regulation of anti-apoptotic molecules. Our in vivo results demonstrate synergistic effects of combination therapy with C646 and proton beam. In BxPC-3 xenograft tumor model, C646 pre-treatment increased proton-induced tumor growth inhibition and apoptotic cell death in tumor tissues. In addition, the combination treatment of C646 with proton beam increased DNA damage and decreased activation of DNA repair pathway compared with proton alone. ConclusionsOur results suggest that the C646 may increase proton- induced apoptosis through the modulation of various pro- and anti- apoptotic molecules in human pancreatic cancer cells. These results provide proof of principle that the inhibition of histone acetylation by HATis can be exploited to develop new proton-sensitizer. Legal entity responsible for the studyThe authors. FundingNational Research Foundation of Korea (NRF). DisclosureAll authors have declared no conflicts of interest.

Vascularization, Oxygenation, and the Effect of Sunitinib Treatment in Pancreatic Ductal Adenocarcinoma Xenografts.

The purpose of the study was to investigate vascularization, oxygenation, and the effect of sunitinib treatment in pancreatic ductal adenocarcinoma (PDAC). BxPC-3 and Capan-2 xenografts grown in dorsal window chambers were used as preclinical models of human PDAC. Tumor angiogenesis and the morphology and function of tumor vascular networks were studied by intravital microscopy, and tumor hypoxia was assessed by immunohistochemistry. The PDAC models differed in vessel distribution and oxygenation, and the differences were induced by the initial tumor angiogenesis. In both models, sunitinib treatment reduced intratumor and peritumor vessel densities by selectively removing small-diameter vessels. Sunitinb treatment resulted in a general decrease in vessel density and scattered hypoxia in BxPC-3 tumors, and depleted most vessels and induced massive hypoxia in central parts of Capan-2 tumors. The study demonstrates that PDAC xenografts can differ in vascularization, and the differences can impact oxygenation and effects of treatment. Neoadjuvant sunitinib treatment is inappropriate in combination with conventional therapy for human PDACs resembling the PDAC xenografts used here, because sunitinib-induced hypoxia can impair the effect of most conventional therapies.

Abstract 3953: FF-10832 combined with immune checkpoint blockade shows favorable anti-tumor activity

Abstract Introduction: We previously reported that FF-10832, a liposomal gemcitabine, exerted anti-tumor activity superior to that of gemcitabine with high stability, long plasma half-life, and preferential tumor accumulation in preclinical tumor models. Beneficial gemcitabine immune-modulating effects in tumor microenvironments have also been reported. We investigated the immune-modulating effects of FF-10832 by evaluating its anti-tumor activities combined with immune checkpoint blockade. Methods: Murine syngeneic tumor models were created by subcutaneous implantation of MBT-2 (bladder) or EMT6 (breast) tumor cells and tumor volume evaluated during and after treatments. Treatments included 2 or 4 mg/kg of FF-10832 or 240 mg/kg of gemcitabine intravenously once a week, and 10 mg/kg of an immune checkpoint inhibitor intraperitoneally twice a week for 3 weeks. Results: In the MBT-2 tumor model, although tumor growth was slightly inhibited by anti-PD-L1 mAb (clone: 10F.9G2) alone, only one out of nine mice showed complete response. Although FF-10832 decreased tumor growth during treatment (3 weeks), remission was incomplete, and tumor growth continued after treatment was discontinued. In contrast, in combination, tumor growth was inhibited even after treatment withdrawal; three out of nine mice achieved complete remission, and median survival improved (vehicle control and anti-PD-L1 mAb: 25 days; FF-10832: 28 days; combination: &amp;gt;49 days). This combination effect was comparable to that of gemcitabine combination. Previously, FF-10832 has also shown anti-tumor effects in a gemcitabine-insensitive BxPC-3 xenograft tumor model, so the potential benefit in combination with checkpoint inhibitors was evaluated in a gemcitabine-insensitive EMT6 tumor model. FF-10832 significantly inhibited tumor growth relative to that of gemcitabine in this model, but all mice showed tumor re-growth after treatment withdrawal. Anti-CTLA-4 mAb (clone: 9H10) alone showed complete response in only one out of eight mice. Strikingly, FF-10832 in combination with anti-CTLA-4 mAb exerted superior anti-tumor activity compared to that with gemcitabine, as shown by tumor regression in all mice and complete regression in seven out of eight mice. Further analysis to clarify the mode of action underlying the combination effect is ongoing now. These results indicate that the effect of immune checkpoint inhibitors is enhanced by FF-10832 regardless of tumor sensitivity to gemcitabine. Conclusion: Although immune checkpoint blockade is thought to be a breakthrough for cancer treatment, only a limited patient population has achieved favorable outcomes. Our results suggested that FF-10832 enhances the anti-tumor activity of immune checkpoint blockade, which could significantly improve treatment outcomes. Citation Format: Tadaaki Ioroi, Takashi Komori, Takeshi Matsumoto, Susumu Shimoyama, Takayuki Kobayashi, Hidetoshi Murao, Shinichi Watanabe, Shinji Hagiwara, Takefumi Hara. FF-10832 combined with immune checkpoint blockade shows favorable anti-tumor activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3953.

Abstract 4823: Elimination of invasive pancreatic cancer cells by p53-activating oncolytic virotherapy as novel precision medicine

Abstract Background: Pancreatic ductal adenocarcinoma (PDAC) is the most lethal disease due to early onset of local recurrence and distant metastasis. Invasive PDAC cells are the main causes of recurrence and metastasis after curative surgery. The precision medicine based on the genetic alterations in the KRAS, p53, CDKN2A, and SMAD4 genes has been recently expected as a promising strategy for the treatment of PDAC patients; however, therapeutic strategy for targeting these genetic alterations in PDAC has not been developed yet. To eliminate p53-inactivated malignant tumor cells, we have developed telomerase-specific replication-competent oncolytic adenovirus OBP-702 that expresses tumor suppressor p53. In this study, we investigated the in vitro and in vivo therapeutic potential of OBP-702 against PDAC cells. Methods: Four human PDAC cell lines (Capan-1, MIA PaCa-2, BxPC-3, Panc-1) with different invasive property were used. The therapeutic effect of OBP-702 and p53-nonexpressing OBP-301 was assessed in the proliferation, migration and invasion abilities of PDAC cells. The underlying mechanism of virus-mediated therapeutic effect was analyzed on the modulation of p53 signaling and KRAS-MAPK signaling. Subcutaneous and orthotopic BxPC-3 xenograft tumor models were used to evaluate the virus-mediated antitumor efficacy. Results: OBP-702 induced antitumor effect in association with autophagy and apoptosis more strongly compared to OBP-301 in PDAC cells through activation of p53 expression. OBP-702 inhibited the migration and invasion properties of PDAC cells more efficiently compared to OBP-301 through suppression of KRAS-ERK1/2 signaling, even when ERK signaling is enhanced by nerves and neurosecretory factors. Similar with OBP-702, treatment with ERK1/2 inhibitor or siRNA significantly reduced migration and invasion abilities. Moreover, OBP-702 significantly suppressed tumor growth in subcutaneous and orthotopic BxPC-3 xenograft tumor models. Conclusions: Our results suggest that OBP-702 is a promising antitumor reagent to eliminate invasive PDAC cells through p53 activation and ERK suppression. Further clinical study is warranted to evaluate the safety and feasibility of OBP-702 as a novel precision medicine based on genetic alterations in KRAS and p53 genes. Citation Format: Takuro Fushimi, Hiroshi Tazawa, Takeshi Koujima, Hiroyuki Araki, Takeyoshi Nishiyama, Satoru Kikuchi, Shinji Kuroda, Ryuichi Yoshida, Hiroyuki Kishimoto, Masahiko Nishizaki, Yasuo Urata, Shunsuke Kagawa, Toshiyoshi Fujiwara. Elimination of invasive pancreatic cancer cells by p53-activating oncolytic virotherapy as novel precision medicine [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4823.

Anti-tumor effect and mechanistic study of elemene on pancreatic carcinoma

BackgroundElemene is an effective anticancer component extracted from Zingiberaceae plants. This work was aimed to evaluate the anti-tumor effect and mechanism actions of elemene on pancreatic carcinoma in vitro and in vivo.MethodsThe anti-proliferation experiment was measured by Methylthiazolyldiphenyl-tetrazolium bromide (MTT) method in the time of 24, 48 and 72 h in three different dosages. The cell cycle was detected by flow cytometer after 12 h treatment. Forty-eight nude mice were subcutaneously xenograft with BxPC-3 pancreatic cancer cells and divided into four groups: Control group and high, medium, low dosage of elemene (20, 40 and 60 mg/kg) treatment groups. Immunoblot and immunohistochemical methods were applied to detect the protein expression of P53 and Bcl-2 in the tumor of pancreatic cancer xenografts. H & E staining was used to detect the histopathological changes in each group.ResultsA significant inhibition effect was observed in the anti-proliferation of BxPC-3 and Panc-1 cells in vitro in the time course of 24, 48 and 72 h with a dose dependent manner. The cell cycle results showed that elemene could arrest pancreatic cancer cells in the S phase after 12 h treatment in BxPC-3 and Panc-1 cell line. The in vivo BxPC-3 xenografts study exhibited that elemene was significantly decreased the tumor size in the high dosage group, compared to control group. And there is no any significant change in body weight of all animals. H&E pathology section result showed that treatment with elemene significantly decreased the inflammation cells and reduced the histopathological changes with a dose-dependent manner. Meanwhile, treatment with elemene significantly up-regulates the protein expression of P53, while down-regulate the protein expression of Bcl-2 in the tumor tissues, respectively. Furthermore, the western blot result showed that treatment with elemene increased the expression of P53 and decreased the expression of Bcl-2, compared with the control group, which is similar to the results of immunohistochemical staining.ConclusionsThis study suggests that elemene has a potential anti pancreatic cancer effect, down-regulation the protein expression of Bcl-2 and up-regulation the protein expression of P53 in a dose dependent manner may be is the anti-tumor mechanism.

A Glypican-1-Targeted and Gemcitabine-Loaded Biocompatible Nanoplatform for Pancreatic Cancer Near-Infrared Fluorescence/Magnetic Resonance Multimodal Imaging and Therapy

Background: Biomarker-targeted nanocarrier holds promise for early diagnosis and effective therapy of cancer. The aim of this work were to design and evaluate glypican-1(GPC1) targeted, gemcitabine (GEM)-loaded multi-functional gold nanocarrier for near-infrared fluorescence (NIRF)/magnetic resonance (MR) imaging and targeted chemotherapy against pancreatic cancer. Methods: GEM-loaded, anti-GPC1 antibody-modified gold nanocages (AuNCs) (GEM@HAuNCs) were prepared, and then conjugated with GPC1 antibody and Gd/Cy7 to create the multi-functional GPC1-GEM@HAuNCs-Cy7/Gd nanoparticles (GPC1-GEM-NPs). The targeting performance and effect on cell viablity of the GEM-GPC1-NPs were examined in vitro using BXPC-3, PANC-1 (GPC1 positive expression) and 293T (GPC1 negative expression) cells. The NIRF/ MR multimodal imaging properties and therapeutic activities of GPC1-GEM-NPs were tested in vivo in BXPC-3 xenograft mouse model. Findings: GPC1- GEM-NPs exhibited good biocompatibility, excellent colloidal stability, and targeting specificity to GPC1 over-expressing cells. Cell viability assays showed stronger growth inhibition of the GPC1-GEM-NPs against GPC1 positive pancreatic cells in comparison with GEM-NPs and GEM. Blood biochemical and histological analyses showed that the in vivo toxicity of GPC1- GEM-NPs was negligible. Both in vitro and in vivo studies demonstrated that GPC1- GEM-NPs can be used as NIRF/MR contrast agent for pancreatic cancer detection. Treatment of xenografted mice with GPC1- GEM-NPs showed a higher tumor inhibitory effect compared with controls. Interpretation: This theranostic nanoplatform provide early diagnostic and effective therapeutic potential for pancreatic cancer. Funding Statement: This work is supported by the National Natural Science Foundation of China (Grant No. 81771899) and Primary Research & Development Plan of Jiangsu Province (BE2017772). Declaration of Interests: The authors have declared that no conflict of interest exists. Ethics Approval Statement: All animal experiments were approved and conducted according to the principles of the Nanjing University of Chinese Medicine Animal Care Committee (SYXK 2014-0001).