Pancreatic Cancer Patient-derived Xenograft Research Articles

Abstract 3121: Therapeutic potential of a HER3 antibody-drug conjugate for the treatment of HER3-expressing cancers

Abstract To address the need for targeted therapy in the treatment of HER3-positive solid tumors, the human HER3 mAb, seribantumab, was evaluated in the context of an antibody-drug conjugate (ADC). As a proof-of-concept, seribantumab was conjugated with a cleavable valine-citrulline linker and monomethyl auristatin E (MMAE) payload via the stochastic cysteine conjugation method to yield HER3-ADC1. A drug-antibody ratio of 4 was selected for preclinical assessment in HER3-expressing models. Methods: HER3-ADC1 was evaluated in vitro and in vivo, with patritumab deruxtecan (patri-DXd) as a comparator. Binding to BT474 breast carcinoma cells (HER3 high; immunohistochemical (IHC) staining intensity 3+) was measured by flow cytometry. Internalization properties were established using seribantumab and patritumab coupled to Fab-AF488 following 4 hours incubation in SK-BR-3 breast adenocarcinoma (HER3 high; IHC 3+) and HCC1569 breast carcinoma (HER3 low; IHC 0-1+) cell lines. In vitro cytotoxicity was evaluated for HER3-ADC1, isotype-MMAE and free MMAE payload as well as patri-DXd, isotype-DXd and free deruxtecan payload in BT474 cells, SK-BR-3 cells and NCI-H446 lung carcinoma (HER3 low; IHC 0-1+) cells. In vivo anti-tumor activity was assessed for HER3-ADC1, isotype-MMAE, patri-DXd and isotype-DXd in patient derived xenograft (PDX) models of pancreatic (HER3 high; IHC 3+) and breast cancer (HER3 low; IHC 0-1+). Results: HER3-ADC1 binding to cancer cells, endocytosis, MMAE release, and inhibition of proliferation were dependent on HER3 expression. In assays investigating antibody internalization, seribantumab displayed similar internalization capacity as patritumab. For both HER3 mAbs, internalization was greater in a HER3 high than a HER3 low cell line. In cytotoxicity assays, HER3-ADC1 and patri-DXd displayed HER3 expression level-dependent cell killing. HER3-ADC1 outperformed patri-DXd in HER3 high cell lines based on in vitro cytotoxicity. HER3-ADC1 and patri-DXd were ineffective against NCI-H466 cells with low HER3 expression. In a PDX model of pancreatic cancer (HER3 high), HER3-ADC1 induced tumor regression, while isotype-MMAE had only a moderate anti-tumor effect. Both patri-DXd and isotype-DXd had moderate anti-tumor effects, indicating a lack of HER3 target dependence. In a PDX model of breast cancer (HER3 low), HER3-ADC1 and isotype-MMAE lacked anti-tumor activity; isotype-DXd had a moderate anti-tumor effect and patri-DXd had a substantial anti-tumor effect. Conclusions: HER3-ADC1 demonstrated target-dependent in vitro cytotoxicity and in vivo anti-tumor activity in a HER3-expressing pancreatic cancer PDX model. Results from in vitro and in vivo studies highlight the promising therapeutic potential of a seribantumab-based ADC for patients with HER3-expressing cancers. Additional results on the optimization and characterization of HER3-ADC1 will be presented. Citation Format: Thomas O'Hare, Jaclyn Cleveland, Valerie M. Jansen, David Dornan. Therapeutic potential of a HER3 antibody-drug conjugate for the treatment of HER3-expressing cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3121.

Abstract A091: The RAF/MEK clamp avutometinib as the backbone of therapy for pancreatic cancer: Novel combinations with standard of care chemotherapy, FAK inhibitors, KRAS G12D inhibitors and/or autophagy inhibitors

Abstract KRAS mutations (mt) occur in up to 98% of pancreatic ductal adenocarcinoma (PDAC) and represent a key initiating event in PDAC carcinogenesis. Therapeutic efforts targeting the RAS/RAF/MEK/ERK (MAPK) pathway with MEK-only inhibitors have been unsuccessful in substantially modifying PDAC prognosis. Thus, novel strategies are needed to overcome putative mechanisms of resistance to MEK inhibition such as focal adhesion kinase (FAK) pathway activation. Another hallmark of PDAC is its high stromal density, which is thought to limit the penetration of cytotoxic drugs and T cells into the tumor and has been shown to be correlated with FAK hyperactivation, altogether supporting co-targeting the MAPK and FAK pathways to achieve deep and durable responses for patients with PDAC. Avutometinib is a RAF/MEK clamp that potently inhibits MEK kinase activity and induces dominant negative RAF-MEK complexes preventing phosphorylation of MEK by ARAF, BRAF and CRAF. This unique mechanism allows avutometinib to block MEK signaling without the compensatory re-activation of MEK that appears to limit the efficacy of MEK-only inhibitors. Preclinically, avutometinib has shown strong anti-proliferative potency across tumor cell lines carrying KRAS mt including PDAC cell lines. Clinically, avutometinib in combination with the FAK inhibitor (FAKi) defactinib received breakthrough therapy designation for the treatment of patients with recurrent low-grade serous ovarian cancer. Here, we tested the combination of avutometinib and FAK inhibition with standard of care chemotherapy in PDAC mouse models. In a KRAS/p53 pancreatic cancer mouse model, we found that whereas the combination of avutometinib + FAKi induced tumor inhibition and increased survival, addition of chemotherapy (gemcitabine + paclitaxel as a surrogate for nab-paclitaxel) to avutometinib/FAKi induced tumor regression. These results support the ongoing clinical study of avutometinib and defactinib in combination with standard of care chemotherapy (gemcitabine/nab-paclitaxel) for patients with front-line metastatic PDAC (NCT05669482). Other combinations being explored preclinically include avutometinib with G12D inhibition (G12Di) in KRAS G12D PDAC, which accounts for 28% of patients with PDAC. In 3D culture, avutometinib was synergistic with the G12Di MRTX1133 in reducing viability of a panel of KRAS G12D PDAC cell lines. In mouse models, avutometinib enhanced anti-tumor efficacy of MRTX1133 in a KRAS G12D pancreatic cancer patient-derived xenograft model. Furthermore, in KRAS mt PDAC cell lines, avutometinib was synergistic with the autophagy inhibitors chloroquine or apilimod. Indeed, dual treatment with avutometinib + MRTX1333 synergistically decreased proliferation and increased autophagic flux in KRAS G12D PDAC cell lines, supporting testing the triple combination of avutometinib + G12Di + autophagy inhibitor in patients with KRAS G12D pancreatic cancers. In conclusion, we show that avutometinib is a unique RAF/MEK clamp with the potential to become the backbone of therapy for pancreatic cancers. Citation Format: Silvia Coma, Xiuting Liu, Noah L. Pieper, Wen-Hsuan Chang, Kirsten L. Bryant, Channing J. Der, David G. DeNardo, Jonathan A. Pachter. The RAF/MEK clamp avutometinib as the backbone of therapy for pancreatic cancer: Novel combinations with standard of care chemotherapy, FAK inhibitors, KRAS G12D inhibitors and/or autophagy inhibitors [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr A091.

Structure-Activity Relationship of FL118 Platform Position 7 Versus Position 9-Derived Compounds and Their Mechanism of Action and Antitumor Activity.

Structurally, FL118 is a camptothecin analogue and possesses exceptional antitumor efficacy against human cancer through a novel mechanism of action (MOA). In this report, we have synthesized and characterized 24 FL118 Position 7-substituted and 24 FL118 Position 9-substituted derivatives. The top compounds were further characterized for their MOA in colorectal cancer (CRC) models using CRC patient-derived xenograft (PDX) models and pancreatic cancer PDX models to evaluate their antitumor activities. Four FL118 Position 7-substituted derivatives showed significantly better antitumor efficacy than the FL118 Position 9-substituted derivatives. The four identified compounds also appeared to have better antitumor activity than their parental platform FL118. Interestingly, RNA-Seq analyses indicated that three of the four compounds exerted antitumor effects via an MOA similar to FL118, which provided an intriguing opportunity for follow-up studies. Extended in vivo studies revealed that FL77-6 (7-(4-ethylphenyl)-FL118), FL77-9 (7-(4-methoxylphenyl)-FL118), and FL77-24 (7-(3, 5-dimethoxyphenyl)-FL118) exhibit potential for further development toward clinical trials.

Development of a Novel CLDN18.2-directed Monoclonal Antibody and Antibody-Drug Conjugate for Treatment of CLDN18.2-Positive Cancers.

Gastric and pancreatic cancers are malignancies of high unmet clinical need. Expression of CLDN18.2 in these cancers, coupled with it's absence from most normal tissues, provides a potential therapeutic window against this target. We present preclinical development and characterization of a novel therapeutic mAb and antibody-drug conjugate (ADC) targeting CLDN18.2. A humanized CLDN18.2 specific mAb, CLDN18.2-307-mAb, was generated through immunization in mice followed by full humanization of the mouse mAb sequences. Antibody clones were screened by flow cytometry for selective binding to membrane bound CLDN18.2. A CLDN18.2-directed ADC (CLDN18.2-307-ADC) was also generated by conjugating MMAE to CLDN18.2 mAb using a cleavable linker. Tissue expression of CLDN18.2 was determined by IHC assay using a CLDN18.2-specific mAb. CLDN18.2-307-mAb binds with high affinity to CLDN18.2-positive (CLDN18.2+) cells and induces antibody-dependent cell-mediated cytotoxicity (ADCC). Treatment with this CLDN18.2-mAb blocked the growth of CLDN18.2+ gastric and pancreas cancer cell line xenograft (CDX) models. Upon binding to the extracellular domain of this target, the CLDN18.2-ADC/CLDN18.2 protein was internalized and subsequently localized to the lysosomal compartment inducing complete and sustained tumor regressions in CLDN18.2+ CDXs and patient-derived pancreatic cancer xenografts (PDX). A screen of human cancer tissues, by IHC, found 58% of gastric, 60% of gastroesophageal junction, and 20% of pancreatic adenocarcinomas to be positive for membrane expression of CLDN18.2. These data support clinical development of the CLDN18.2-307-mAb and CLDN18.2-307-ADC for treatment of CLDN18.2+ cancers. Both are now being investigated in phase I clinical studies.

Species-Deconvolved Proteomics for In Situ Investigation of Tumor-Stroma Interactions after Treatment of Pancreatic Cancer Patient-Derived Xenografts with Combined Gemcitabine and Paclitaxel.

Tumor-stroma interactions are critical in pancreatic ductal adenocarcinoma (PDAC) progression and therapeutics. Patient-derived xenograft (PDX) models recapitulate tumor-stroma interactions, but the conventional antibody-based immunoassay is inadequate to discriminate tumor and stromal proteins. Here, we describe a species-deconvolved proteomics approach embedded in IonStar that can unambiguously quantify the tumor (human-derived) and stromal (mouse-derived) proteins in PDX samples, enabling unbiased investigation of tumor and stromal proteomes with excellent quantitative reproducibility. With this strategy, we studied tumor-stroma interactions in PDAC PDXs that responded differently to Gemcitabine combined with nab-Paclitaxel (GEM+PTX) treatment. By analyzing 48 PDX animals 24 h/192 h after treatment with/without GEM+PTX, we quantified 7262 species-specific proteins under stringent cutoff criteria, with high reproducibility. For the PDX sensitive to GEM+PTX, the drug-dysregulated proteins in tumor cells were involved in suppressed oxidative phosphorylation and the TCA cycle, and in the stroma, inhibition of glycolytic activity was predominant, suggesting a relieved reverse Warburg effect by the treatment. In GEM+PTX-resistant PDXs, protein changes suggested extracellular matrix deposition and activation of tumor cell proliferation. Key findings were validated by immunohistochemistry (IHC). Overall, this approach provides a species-deconvolved proteomic platform that could advance cancer therapeutic studies by enabling unbiased exploration of tumor-stroma interactions in the large number of PDX samples required for such investigations.

18F]AlF-NOTA-ADH-1: A new PET molecular radiotracer for imaging of N-cadherin-positive tumors.

The cell adhesion molecule (CAM) N-cadherin has become an important target for tumor therapy. The N-cadherin antagonist, ADH-1, exerts significant antitumor activity against N-cadherin-expressing cancers. In this study, [18F]AlF-NOTA-ADH-1 was radiosynthesized. An in vitro cell binding test was performed, and the biodistribution and micro-PET imaging of the probe targeting N-cadherin were also studied in vivo. Radiolabeling of ADH-1 with [18F]AlF achieved a yield of up to 30% (not decay-corrected) with a radiochemical purity of >97%. The cell uptake study showed that Cy3-ADH-1 binds to SW480 cells but weakly binds to BXPC3 cells in the same concentration range. The biodistribution results demonstrated that [18F]AlF-NOTA-ADH-1 had a good tumor/muscle ratio (8.70±2.68) in patient-derived xenograft (PDX) tumor xenografts but a lower tumor/muscle ratio (1.91±0.69) in SW480 tumor xenografts and lowest tumor/muscle ratio (0.96±0.32) in BXPC3 tumor xenografts at 1 h post-injection (p.i.) These findings were in accordance with the immunohistochemistry results. The micro PET imaging results revealed good [18F]AlF-NOTA-ADH-1 tumor uptake in pancreatic cancer PDX xenografts with strong positive N-calcium expression, while lower tumor uptake in SW480 xenografts with positive expression of N-cadherin, and significantly lower tumor uptake in BXPC3 xenografts with low expression of N-cadherin, which was consistent with the biodistribution and immunohistochemistry results. The N-cadherin-specific binding of [18F]AlF-NOTA-ADH-1 was further verified by a blocking experiment involving coinjection of a non radiolabeled ADH-1 peptide, resulting in a significant reduction in tumor uptake in PDX xenografts and SW480 tumor. [18F]AlF-NOTA-ADH-1 was successfully radiosynthesized, and Cy3-ADH-1 showed favorable N-cadherin-specific targeting ability by in vitro data. The biodistribution and microPET imaging of the probe further showed that [18F]AlF-NOTA-ADH-1 could discern different expressions of N-cadherin in tumors. Collectively, the findings demonstrated the potential of [18F]AlF-NOTA-ADH-1 as a PET imaging probe for non-invasive evaluation of the N-cadherin expression in tumors.

Biological evaluation of novel gemcitabine analog in patient-derived xenograft models of pancreatic cancer

Gemcitabine (Gem) has been a standard first-line drug for pancreatic cancer (PCa) treatment; however, Gem's rapid metabolism and systemic instability (short half-life) limit its clinical outcome. The objective of this study was to modify Gem into a more stable form called 4-(N)-stearoyl-gemcitabine (4NSG) and evaluate its therapeutic efficacy in patient-derived xenograft (PDX) models from PCa of Black and White patients.Methods 4NSG was synthesized and characterized using nuclear magnetic resonance (NMR), elemental analysis, and high-performance liquid chromatography (HPLC). 4NSG-loaded solid lipid nanoparticles (4NSG-SLN) were developed using the cold homogenization technique and characterized. Patient-derived pancreatic cancer cell lines labeled Black (PPCL-192, PPCL-135) and White (PPCL-46, PPCL-68) were used to assess the in vitro anticancer activity of 4NSG-SLN. Pharmacokinetics (PK) and tumor efficacy studies were conducted using PDX mouse models bearing tumors from Black and White PCa patients.Results 4NSG was significantly stable in liver microsomal solution. The effective mean particle size (hydrodynamic diameter) of 4NSG-SLN was 82 ± 6.7 nm, and the half maximal inhibitory concentration (IC50) values of 4NSG-SLN treated PPCL-192 cells (9 ± 1.1 µM); PPCL-135 (11 ± 1.3 µM); PPCL-46 (12 ± 2.1) and PPCL-68 equaled to 22 ± 2.6 were found to be significantly lower compared to Gem treated PPCL-192 (57 ± 1.5 µM); PPCL-135 (56 ± 1.5 µM); PPCL-46 (56 ± 1.8 µM) and PPCL-68 (57 ± 2.4 µM) cells. The area under the curve (AUC), half-life, and pharmacokinetic clearance parameters for 4NSG-SLN were 3–fourfold higher than that of GemHCl. For in-vivo studies, 4NSG-SLN exhibited a two-fold decrease in tumor growth compared with GemHCl in PDX mice bearing Black and White PCa tumors.Conclusion 4NSG-SLN significantly improved the Gem's pharmacokinetic profile, enhanced Gem's systemic stability increased its antitumor efficacy in PCa PDX mice bearing Black and White patient tumors.

O011 Investigating the effect of supraphysiological levels of calcium on irreversible electroporation (IRE) of pancreatic cancer cell lines

Abstract Introduction Pancreatic cancer remains difficult to treat with a 5-year survival of 2%. Loco-regional therapies are being introduced such as Irreversible Electroporation (IRE); a non-thermal ablative therapy thought to cause cell death by increasing the cell membrane potential resulting in the formation of aqueous pores, leading to cell death. Mitochondrial changes have previously been reported to be a key driver of apoptosis and calcium has been implicated in this process. We aimed to study the influence of calcium in combination with IRE in pancreatic cancer. Methods Immortalised pancreatic cancer cell line, PANC-1, and pancreatic cancer patient-derived xenograft cells (PDX 185) were exposed to calcium (Ca2+) and IRE monotherapy and in combination. Cells were trypsinised and electroporated in 4mm cuvettes with a BTX generator (Harvard Apparatus). Cell proliferation and viability were analysed, and depolarised mitochondria were evaluated by TMRE and analysed using flow cytometry. Results IRE combined with calcium was more cytotoxic compared to monotherapy with cell proliferation assays demonstrating a statistically significant reduction in proliferation (p<0.0001) across both cell lines. Interestingly, mitochondrial membrane potential analyses demonstrated a depolarising effect only in cells exposed to both calcium and IRE suggesting a mitochondrial mediated effect in cell death in these groups. Conclusion Calcium in combination with IRE may act to potentiate cell death through depolarisation of the mitochondrial membrane. Future work will involve understanding the impact of IRE in combination with calcium in 3D models, allowing the evaluation of molecular cell death mechanisms.

Abstract 816: Biological evaluation of novel stearoyl gemcitabine nanoparticles in primary pancreatic cancer cells and PDX mice tumor models

Abstract Purpose: This study aimed to improve the systemic stability and enhance the anticancer activity of gemcitabine (Gem) by modifying Gem with stearic acid to form 4-N-stearoylGem (4NSG). The 4NSG compound was formulated into solid lipid nanoparticles and tested against patient-derived pancreatic cancer (PCa) cell lines and patient-derived xenograft (PDX) mice bearing subcutaneous tumors. Methods: Gem was modified by linking the 4-amino group of Gem and stearoyl acyl derivative to form 4-(N)-stearoyl-gemcitabine (4NSG). 4NSG was characterized using high-performance liquid chromatography (HPLC), nuclear magnetic resonance (NMR), and elemental analysis. 4NSG was developed into solid lipid nanoparticles (4NSG-SLN) and characterized using a particle size analyzer. Patient-derived primary PCa cells (PPCL-46 and PPCL-68) were treated with GemHCl and 4NSG-SLN. Cytotoxicity, cell migration, and cell-cycle studies were performed to determine the effectiveness of 4NSG-SLN against primary PCa cell lines. We performed antitumor efficacy testing using GemHCl and 4NSG-SLN in PDX mice bearing subcutaneous pancreatic tumors. Results: Analysis of the H-NMR spectra displayed an amide bond at 10.88 ppm, confirming the conjugated bond between the 4-amino group of Gem and stearic acid. The purity of 4NSG was 99.8%. The hydrodynamic diameter (particle size) of 4NSG-SLN was 82±2.3nm, while the blank nanoparticle (SLN) was found to be 35 ± 4.3 nm. The half-maximal inhibitory concentration of 4NSG-SLN-treated PPCL-46 (IC50 =12 ± 2.1µM) and PPCL-68 cultures (IC50 =22 ± 2.6µM) showed higher cytotoxic activity compared with GemHCl-treated PPCL-46 and PPCL-68 cultures respectively (IC50 = 56 ± 2.4 µM, IC50 = 57 ± 1.5 µM, p-value: p<0.001). Our migration study showed 4NSG-SLN treated PPCL-46 cells at 5μΜ concentrations significantly reduced cell mobility towards the wound area with (28 ± 3.5) number of cells migrated compared to GemHCl treatment which showed (120 ± 3.8). The cell-cycle analysis showed that 4NSG-SLN treated PPCL-46 cells at 5μΜ concentration had a higher G1 population (78.25%) than GemHCl (72.75%). 4NSG-SLN may have triggered apoptosis in PPCL-46 cells (2.45%) at 20μΜ in the S and G2/M phases compared to GemHCl treatments which showed a higher cell population of (9.54%).4NSG-SLN treated PDX mice exhibited a two-fold decrease in tumor growth compared to GemHCl-treated PDX mice bearing tumors. Conclusion: This study reveals that 4NSG-SLNs may potentially prolong Gem's systemic circulation, increase its bioavailability, and, most importantly, enhance the therapeutic efficacy of Gem in the treatment of PCa. Citation Format: Esther K. Frimpong, Andriana Inkoom, Nkafu B. Ndemazie, Raviteja Bulusu, Joy C. Okoro, Bo Han, Jose Trevino, Edward Agyare. Biological evaluation of novel stearoyl gemcitabine nanoparticles in primary pancreatic cancer cells and PDX mice tumor models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 816.

MP04-13 IMMUNOPHENOTYPE ANALYSIS OF TUMOR-INFILTRATING IMMUNE CELLS TO ELUCIDATE THE MECHANISM OF ANTITUMOR EFFECT OF IL-7 AND CCL19 PRODUCING CAR-T CELLS AGAINST SOLID CANCER

You have accessJournal of UrologyCME1 Apr 2023MP04-13 IMMUNOPHENOTYPE ANALYSIS OF TUMOR-INFILTRATING IMMUNE CELLS TO ELUCIDATE THE MECHANISM OF ANTITUMOR EFFECT OF IL-7 AND CCL19 PRODUCING CAR-T CELLS AGAINST SOLID CANCER Shunsuke Goto, Koji Tamada, and Masatoshi Eto Shunsuke GotoShunsuke Goto More articles by this author , Koji TamadaKoji Tamada More articles by this author , and Masatoshi EtoMasatoshi Eto More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003215.13AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Chimeric antigen receptor (CAR)-T cell therapy against solid cancer is challenging. We reported that mouse CAR-T cells modified to produce interleukin- (IL)-7 and CCL19 (7×19 CAR-T) enhance the therapeutic efficacy against solid tumors by recruiting not only administered CAR-T cells but also endogenous T cells and DCs into tumors and inducing long-term antitumor memory responses. Furthermore, we reported that human 7×19 CAR-T cells show enhanced antitumor efficacy against patient-derived xenograft (PDX) tumor expressing mesothelin, for which 10% of renal cell carcinoma are positive. In this research, we explore the mechanism of antitumor activity of 7×19 CAR-T cells and object to prevail the difference between conventional CAR-T cells (Conv.) and 7×19, especially focusing on the immunophenotype of tumor-infiltrating lymphocytes (TILs). METHODS: We produced a novel anti-human mesothelin 7×19 CAR-T cells, then assessed the antitumor activity against solid tumor in vivo using a malignant mesothelioma orthotopic tumor and a pancreatic cancer PDX models. Tumor was resected after the administration of Conv. or 7×19 CAR-T cells and analyzed TILs by flowcytometry focusing on the number, exhaustion marker, phenotype and cytokine producing ability. RESULTS: 7×19 CAR-T cells showed superior antitumor efficacy compared to Conv. CAR-T cells in the orthotopic and PDX tumor models. In the PDX treatment model, TIL analysis revealed that not only CAR-T cells but also non-CAR-T cells were infiltrated effectively into tumors by 7×19 treatment compared to Conv. Exhaustion marker expressions such as PD-1 and TIGIT were significantly downregulated in 7×19 than Conv. The examination of the ability of TILs to express cytokines and effector molecules showed that significant increases of TNF-α and Granzyme B, as well as a trend of increased IFN-γ in 7×19 CAR-T cells. The analysis of splenocyte after the treatment revealed that 7×19 CAR-T cells maintained their effector memory phenotype. CONCLUSIONS: It was found that 7×19 CAR-T cells showed enhanced antitumor potential by the maintenance of their effector functions. These data support the foresight of this therapy as a treatment option for patients with mesothelin-positive renal cell carcinoma. Source of Funding: Practical Research for Innovative Cancer Control, and Project for Cancer Research and Therapeutic Evolution (P-CREATE) 16770206 by Japan Agency for Medical Research and Development (AMED) © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e38 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Shunsuke Goto More articles by this author Koji Tamada More articles by this author Masatoshi Eto More articles by this author Expand All Advertisement PDF downloadLoading ...

Genetics and Therapeutic Responses to Tumor-infiltrating Lymphocyte Therapy of Pancreatic Cancer Patient-derived Xenograft Models

Genetics and Therapeutic Responses to Tumor-infiltrating Lymphocyte Therapy of Pancreatic Cancer Patient-derived Xenograft Models

Critical evaluation of an autologous peripheral blood mononuclear cell-based humanized cancer model.

The use of humanized mouse models for oncology is rapidly expanding. Autologous patient-derived systems are particularly attractive as they can model the human cancer's heterogeneity and immune microenvironment. In this study, we developed an autologous humanized mouse cancer model by engrafting NSG mice with patient-derived xenografts and infused matched peripheral blood mononuclear cells (PBMCs). We first defined the time course of xenogeneic graft-versus-host-disease (xGVHD) and determined that only minimal xGVHD was observed for up to 8 weeks. Next, colorectal and pancreatic cancer patient-derived xenograft bearing NSG mice were infused with 5x106 human PBMCS for development of the humanized cancer models (iPDX). Early after infusion of human PBMCs, iPDX mice demonstrated engraftment of human CD4+ and CD8+ T cells in the blood of both colorectal and pancreatic cancer patient-derived models that persisted for up to 8 weeks. At the end of the experiment, iPDX xenografts maintained the features of the primary human tumor including tumor grade and cell type. The iPDX tumors demonstrated infiltration of human CD3+ cells with high PD-1 expression although we observed significant intra and inter- model variability. In summary, the iPDX models reproduced key features of the corresponding human tumor. The observed variability and high PD-1 expression are important considerations that need to be addressed in order to develop a reproducible model system.

Abstract 1548: Examination of miRNA dysregulation in pancreatic cancer PDX models with parallel proteomic and transcriptomic investigations

Abstract Introduction: Pancreatic cancer is a high-fatality cancer. An estimated 57,600 new cases were diagnosed in the United States in 2020 with a corresponding 47,050 deaths. Despite improvements in the molecular understanding of pancreatic cancer, the prognosis for this disease has not improved in nearly 4 decades, largely because of the lack of early stage disease detection and limited efficiency of systemic therapies. MicroRNAs (miRNAs) are a class of non-coding RNAs approximately 22 nucleotides (nt) in length that regulate gene expression. in recent years, the dysregulation of miRNAs has been implicated in the development of various human cancers. Methods: Parallel analysis of global mRNA, miRNA and protein expression was conducted on pancreatic cancer patient-derived xenografts. The data from this analysis of these F1 tumours, was used to refine/identify miRNA targets of interest to generate 2 differential expression (DE) lists for miRNA; 1) Tumour vs Normal and 2) Tumour vs F1. Predictive software was used to identify targets and pathways of the DE priority miRNAs. These targets were further refined by integrating the targets with the global mRNA and proteomic profiling (unpublished data) performed on the same clinical samples. Alteration of cancer phenotype was investigated using overexpression and/or sponge vectors for each miRNA. Results: Analysis generated a list of priority miRNAs that had inverse interactions with predicted protein targets and were shown to be differentially expressed in our proteomic data. From 39 miRNAs investigated from the priority list, 7 miRNAs targeted 2 or more proteins across the 2 comparisons. These miRNAs - miRs-2467, -4742, -509, -615-3p, -4534, -222 and 2-06 when altered in expression MIA PaCa-2 and PANC-1 pancreatic cancer cells resulted in modulated cell proliferation, colony forming ability and anoikis resistance. Studies are ongoing on the changes to the chemotherapy and radiosensitivity of each cell line in response to changing these miRNA levels. Conclusions: The study identified miRNAs which displayed increased expression between normal and tumour and again increased between tumour and F1. From this list using predicative software and integration with additional global analysis of mRNA and Protein expression a priority list of 7 miRNAs were identified. The core concept in this study was to use differential expression of miRNAs as a tool to further unravel the biology of human pancreatic cancer, in the hope of uncovering new targets or vulnerabilities which might be exploited therapeutically or finding new bio markers which might contribute to better or earlier diagnosis. Citation Format: Taylor-Jade Allen. Examination of miRNA dysregulation in pancreatic cancer PDX models with parallel proteomic and transcriptomic investigations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1548.

Abstract 5824: Fully-humanized, bioengineered miR-1291 modulates key nutrient transport and metabolism to control tumor metabolism

Abstract Tumor progression is accompanied by metabolic reprogramming to allow carcinoma cells to access essential nutrients under the tumor microenvironment. To meet energetic and material needs for high-rate proliferation and tumorigenesis, cancer cells generally rely more heavily on vital nutrients (e.g., glucose, amino acids, and vitamins) than normal cells. Therefore, controlling nutrient transport and metabolism in cancer cells is a promising therapeutic strategy. Our previous studies have suggested that microRNA-1291-5p (miR-1291-5p or miR-1291) is downregulated in pancreatic cancer (PC) cells and acts as a tumor suppressor. The current study is to delineate the role of miR-1291 in the control of PC cell metabolism and develop miR-1291 therapy. We first employed our newly-invented RNA bioengineering technology to achieve high-level and large-scale, in vivo fermentation production of a fully-humanized biologic miR-1291 agent at a high degree of homogeneity (> 4 mg BioRNA per liter of culture and > 97% purity). RNA sequencing studies revealed that BioRNA/miR-1291 was selectively processed to target miR-1291-5p to modulate the transcriptome in PC cells. Proteomics study showed that a number of proteins were reduced remarkably in AsPC-1 cells by biologic miR-1291-5p, among which PNPO, an enzyme that mediates the synthesis of essential nutrient vitamin B6 (VB6), was revealed as a leading target of miR-1291-5p. Besides PNPO and the previously-reported glucose transporter SLC2A1/GLUT1, amino acid transporter SLC7A5/LAT1 was verified as a direct target for miR-1291-5p. Downregulation of PNPO, LAT1, GLUT1 protein expression by biological miR-1291 led to sharp alteration of homeostasis of glucose, amino acids, and VB6 in human PC cells, and subsequent suppression of glycolysis capacity and mitochondrial function as well as the increase of oxidative stress. Combination treatment with bioengineered miR-1291 and 5-FU exerted strong synergism in the inhibition of PC cell and organoid proliferation in vitro. In addition, miR-1291 mono- and combination therapy were effective to control tumor growth in pancreatic cancer patient-derived xenograft (PDX) mouse models. These results demonstrate a critical role for miR-1291 in the regulation of PC cell metabolism which provides insight in developing new therapeutic strategies for PC. Citation Format: Mei-Juan Tu, Frank J. Gonzalez, Ai-Ming Yu. Fully-humanized, bioengineered miR-1291 modulates key nutrient transport and metabolism to control tumor metabolism [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5824.

Abstract 5567: hYP218 CAR-T cells targeting a membrane-proximal epitope of mesothelin are highly effective against mesothelin expressing cancers

Abstract Background and Hypothesis: Mesothelin is an attractive target for CAR-T cell therapy since it is highly expressed in many solid tumors but has limited expression on normal human tissues. However, anti-mesothelin CAR-T cells have shown limited efficacy in patients. Since most antibodies to mesothelin bind the membrane distal epitopes we generated antibodies that bind mesothelin in a membrane-proximal region. We hypothesize that CAR-T cells recognizing a membrane proximal epitope on mesothelin will be more efficacious. Study Design: We developed CAR-T cells targeting a membrane-distal (SS1CAR-T) and a membrane-proximal epitope (hYP218 CAR-T) of mesothelin. Using mesothelin-expressing cancer cell lines, anti-tumor efficacy of CAR-T cells was assessed in vitro by measuring direct killing of tumor cells by the CAR-T cells and quantifying cytokine-release by the CAR-T cells. In vivo anti-tumor efficacy of CAR-T was determined in NSG mice using three different solid tumor models of high-mesothelin expression: ovarian cancer (OVCAR-8), pancreatic cancer (KLM-1), and mesothelioma patient-derived xenograft (NCI-Meso63). Blood and tumor infiltration of CAR-T cells in vivo was determined via flow cytometry analysis. Results: Upon co-culture with tumor cells with high mesothelin expression (A431-H9, OVCAR-8, KLM-1, H226, NCI-Meso63, NCI-Meso29), hYP218 CAR-T cells killed cancer cells more efficiently than SS1 CAR-T cells, with a 2-4-fold lower ET50 value (Effector to Target ratio for 50% killing of tumor cells). Additionally, compared to SS1 CAR-T cells, hYP218 CAR-T cells secreted significantly higher level of cytokines (IL-2, TNFα, IFNγ) in a co-culture with tumor cells. In three solid tumor models, single treatment of tumor-bearing mice with hYP218 CAR-T cells lead to improved tumor response and survival compared to SS1 CAR-T, with complete regression of OVCAR-8 and NCI-Meso63 tumors. Compared to SS1 CAR-T cells, higher hYP218 CAR-T cell infiltration was detected both in the blood and tumors isolated from hYP218 CAR-T treated mice seven days post-treatment. Conclusion: We demonstrate that hYP218 CAR-T cells targeting a membrane-proximal epitope of mesothelin are more potent than similar CAR-T cells targeting a membrane distal epitope. Increased accumulation of hYP218 CAR-T cells in the tumor may explain their increased efficacy. These results support its clinical development to treat patients with mesothelin expressing cancers. Citation Format: Sakshi Tomar, Jingli Zhang, Nan Li, Ira Pastan, Mitchell Ho, Raffit Hassan. hYP218 CAR-T cells targeting a membrane-proximal epitope of mesothelin are highly effective against mesothelin expressing cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5567.

Lipocalin 2 may be a key factor regulating the chemosensitivity of pancreatic cancer to gemcitabine

Owing to the high heterogeneity of pancreatic cancer, patient-derived xenografts (PDX) can compensate for the defects of cell line-derived xenografts (CDX) and also better preserve the heterogeneity and tumor microenvironment of primary tumors. Further, gemcitabine, which is used for the treatment of various cancers, is prone to tumor drug resistance, and this limits its sustained efficacy. Therefore, in this study, our objective was to screen appropriate individual therapeutic drugs for pancreatic cancer. To this end, we established pancreatic cancer PDX models from different patients and screened gemcitabine sensitivity regulatory molecules via high-throughput transcriptome sequencing and bioinformatics analysis. Based on the results obtained, gemcitabine was identified as the most suitable chemotherapeutic drug in a variety of PDX models. Additionally, our results indicated that Lipocalin 2 (LCN 2) may play an important role in the sensitivity of pancreatic cancer to gemcitabine treatment. Thus, the study provides a new potential intervention target for the treatment of pancreatic cancer in clinical practice.

Development of Highly Effective Anti-Mesothelin hYP218 Chimeric Antigen Receptor T Cells With Increased Tumor Infiltration and Persistence for Treating Solid Tumors.

Mesothelin targeting CAR T cells have limited activity in patients. In this study, we sought to determine if efficacy of anti-mesothelin CAR T cells is dependent on the mesothelin epitopes that are recognized by them. To do so, we developed hYP218 (against membrane-proximal epitope) and SS1 (against membrane-distal epitope) CAR T cells. Their efficacy was assessed in vitro using mesothelin-positive tumor cell lines and in vivo in NSG mice with mesothelin-expressing ovarian cancer (OVCAR-8), pancreatic cancer (KLM-1), and mesothelioma patient-derived (NCI-Meso63) tumor xenografts. Persistence and tumor infiltration of CAR T cells was determined using flow cytometry. hYP218 CAR T cells killed cancer cells more efficiently than SS1 CAR T cells, with a two- to fourfold lower ET50 value (effector-to-target ratio for 50% killing of tumor cells). In mice with established tumors, single intravenous administration of hYP218 CAR T cells lead to improved tumor response and survival compared with SS1 CAR T cells, with complete regression of OVCAR-8 and NCI-Meso63 tumors. Compared with SS1 CAR T cells, there was increased peripheral blood expansion, persistence, and tumor infiltration of hYP218 CAR T cells in the KLM-1 tumor model. Persistence of hYP218 CAR T cells in treated mice led to antitumor immunity when rechallenged with KLM-1 tumor cells. Our results show that hYP218 CAR T cells, targeting mesothelin epitope close to cell membrane, are very effective against mesothelin-positive tumors and are associated with increased persistence and tumor infiltration. These results support its clinical development to treat patients with mesothelin-expressing cancers.

Anticancer effect of locally applicable aptamer-conjugated gemcitabine-loaded atelocollagen patch in pancreatic cancer patient-derived xenograft models.

We investigated the anticancer effect of the aptamer‐conjugated gemcitabine‐loaded atelocollagen patch in a pancreatic cancer patient–derived xenograft (PDX) model to propose a future potential adjuvant surgical strategy during curative pancreatic resection for pancreatic cancer. A pancreatic cancer PDX model was established. Animals were grouped randomly into a no‐treatment control group; treatment group treated with intraperitoneal gemcitabine injection (IP‐GEM) or aptamer‐conjugated gemcitabine (APT:GEM); and transplant with three kinds of patches: atelocollagen‐aptamer‐gemcitabine (patch I), atelocollagen‐inactive aptamer‐gemcitabine (patch II), and atelocollagen‐gemcitabine (patch III). Tumor volumes and response were evaluated based on histological analysis by H&E staining and Immunohistochemistry (IHC) was performed. Anticancer therapy–related toxicity was evaluated by hematologic findings. The patch I group showed the most significant reduction of tumor growth rate, compared with the no‐treatment group (p < 0.05). However, other treatment groups were not found to show significant reduction in tumor growth rate (0.05 < p < 0.1). There was no microscopic evidence suggesting potential toxicity, such as inflammation, nor necrotic changes in liver, lung, kidney, and spleen tissue. In addition, no leukopenia, anemia, or neutropenia was observed in the patch I group. This implantable aptamer‐drug conjugate system is thought to be a new surgical strategy to augment the oncologic significance of margin‐negative resection in treating pancreatic cancer in near future.

Proteolytic pan-RAS Cleavage Leads to Tumor Regression in Patient-derived Pancreatic Cancer Xenografts.

The lack of effective RAS inhibition represents a major unmet medical need in the treatment of pancreatic ductal adenocarcinoma (PDAC). Here, we investigate the anticancer activity of RRSP-DTB, an engineered biologic that cleaves the Switch I of all RAS isoforms, in KRAS-mutant PDAC cell lines and patient-derived xenografts (PDX). We first demonstrate that RRSP-DTB effectively engages RAS and impacts downstream ERK signaling in multiple KRAS-mutant PDAC cell lines inhibiting cell proliferation at picomolar concentrations. We next tested RRSP-DTB in immunodeficient mice bearing KRAS-mutant PDAC PDXs. Treatment with RRSP-DTB led to ≥95% tumor regression after 29 days. Residual tumors exhibited disrupted tissue architecture, increased fibrosis and fewer proliferating cells compared with controls. Intratumoral levels of phospho-ERK were also significantly lower, indicating in vivo target engagement. Importantly, tumors that started to regrow without RRSP-DTB shrank when treatment resumed, demonstrating resistance to RRSP-DTB had not developed. Tracking persistence of the toxin activity following intraperitoneal injection showed that RRSP-DTB is active in sera from immunocompetent mice for at least 1 hour, but absent after 16 hours, justifying use of daily dosing. Overall, we report that RRSP-DTB strongly regresses hard-to-treat KRAS-mutant PDX models of pancreatic cancer, warranting further development of this pan-RAS biologic for the management of RAS-addicted tumors.

A Pancreatic Cancer Patient-Derived Xenograft Model for Adoptive Immunotherapy Using Autologous Tumor-Infiltrating Lymphocytes

A Pancreatic Cancer Patient-Derived Xenograft Model for Adoptive Immunotherapy Using Autologous Tumor-Infiltrating Lymphocytes