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Background: Systemic disease control is the principle driver of prognosis in patients with pancreatic cancer (PDAC). Currently, there are no predictive biomarkers of clinical response to guide selection of an optimal treatment approach. Our work explores the logistics and feasibility of generating patient tumor-derived models to serve as biomarkers. Methods: Patients with borderline resectable or locally advanced PDAC enrolled to participate in a multi-institutional prospective, randomized, trial were eligible. Pretreatment core needle biopsies from diagnostic endoscopy were shipped via commercial post to a centralized organoid laboratory. Patient-specific organoids (PDO) were established as previously described by our group. Putative predictive biomarkers of clinical treatment response, including whole-exome sequencing (WES), RNA-sequencing, and pharmacotyping (chemotherapeutic sensitivity testing) were performed prospectively. Putative predictive biomarkers will be compared to clinical outcomes when approved by the trial's Data Safety Monitoring Board. Results: Approximately 40% (42/98) of patients participated in tissue acquisition for organoid development. Culture maturation can be described in three relevant phases: establishment, expansion, and characterization. The establishment phase describes the emergence of viable ductal organoid development in the setting of a culture being cleared of other components of the tumor microenvironment. During these first 3-5 passages, there are numerous potential pitfalls, including technical and logistical challenges. Out of 42, 26 (62%) PDOs were successfully established. Notably, the establishment phase appears to be an area of the greatest improvement, as the rates of successful establishment improve while the collaborative team builds experience. Expansion involves biomass accumulation in clean culture. Of those that succeeded in establishment, 77% (20/26) entered in expansion phase. We can currently report 15 PDOs having reached the characterization phase (WES, RNA-seq with subtyping, and/or pharmacotyping). Based on RNA expression profile, 10/14 were classified as classical and 4 as basal-like subtype. Mean time to pharmacotyping was 179 days. The PDOs showed a variable population distribution of sensitivity to standard-of-care chemotherapeutics (gemcitabine, paclitaxel, irinotecan, 5-FU, oxaliplatin). Conclusions: Developing a living organoid biobank from PDAC biopsies in a multi-center trial setting is a tractable approach in precision medicine initiatives. Amongst a collaborative team of physician scientists, the establishment phase is critical. PDOs from pre-treatment biopsies can be individually characterized within a clinically relevant time frame and unique tumor-specific ex vivo-derived therapeutic sensitivities can be assessed. Citation Format: Toni T. Seppälä, Jacquelyn W. Zimmerman, Noah Rozich, Alex Blair, Ammar Javed, John L. Cameron, William R. Burns, Jin He, David Tuveson, Christopher L. Wolfgang, David P. Ryan, Alec Kimmelman, Joseph M. Herman, Wells Messersmith, Theodore S. Hong, David T. Ting, Richard Andrew Burkhart. Patient-derived organoids may facilitate precision medicine in pancreatic cancer: Demonstrating feasibility in the context of a multi-center clinical trial [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr LB-011.

Bispecific T cell engager (BiTE®) antibody constructs are designed to redirect T cells to induce lysis of tumor cells through simultaneous binding to CD3 on T cells and to a tumor associated antigen. BiTE® antibody constructs have previously been shown to be effective at depleting blood and tissue targets in B cell malignancies, suggesting a therapeutic potential for BiTE® antibody constructs in solid tumors. Pancreatic ductal adenocarcinoma (PDA) is a devastating malignant disease with a dismal prognosis even with currently available therapies. The tumor differentiation antigen mesothelin (MSLN) is highly expressed in over 80% of pancreatic tumors. Expression of MSLN is restricted in normal tissue implicating MSLN as an attractive target for BiTE® targeted therapy in PDA. In this study, we report the preclinical characterization of an anti-MSLN/CD3 BiTE® antibody construct with half-life extension (MSLN HLE BiTE®) in xenograft models of PDA. Previously the MSLN HLE BiTE® was found to bind CD3 and MSLN with low nanomolar affinity and have low picomolar cytotoxic activity against MSLN-positive cells in vitro. Moreover, the MSLN HLE BiTE® can mediate redirected lysis of cancer cell lines resistant to chemotherapy. Our study examines the efficacy of the highly potent and specific MSLN HLE BiTE® antibody construct in vivo by utilizing the orthotopic implantation of human AsPC-1 luciferase expressing PDA cells in immunocompromised NOD-Prkdcscid IL2rgnull (NSG) mice. We found that mice receiving the MSLN HLE BiTE® antibody construct survived significantly longer and had significantly reduced tumor burden comparing to control mice. Moreover, tumor bearing mice were given fluorescently labeled human T cells to measure T cell localization. Using live animal fluorescent imaging, we found mice treated with the MLSN HLE BiTE® antibody construct had significantly increased T cell localization and retention to the PDA tumor area compared to control. Mice receiving orthotopic implantation of MSLN knock out human AsPC-1 tumors treated with the MSLN HLE BiTE® did not show increased survival, reduction of tumor burden or increased T cell localization, confirming the BiTE® specificity for the tumor target MSLN. Furthermore, using the KPC mouse model of PDA, which develops cancer spontaneously upon conditional pancreatic expression of KRASG12D and TP53R172H mutations, we tested if a mouse-specific surrogate MSLN HLE BiTE® antibody construct could localize to a naturally formed dense tumor microenvironment. We found that mice treated with a murine MSLN HLE BiTE® labeled with CW800 demonstrated strong localization of the MSLN HLE BiTE® through the dense microenvironment to the PDA tumor. Taken together, these results suggest that a MSLN HLE BiTE® may be a unique targeted immunotherapy for PDA patients. Citation Format: Noelle R. Jurcak, MacKenzie Zarecki, Fei Lee, Noah Rozich, Stephen Muth, Elizabeth Jaffee, Sabine Stienen, Julie Bailis, Lei Zheng. Evaluation of mesothelin BiTE® antibody constructs in models of pancreatic ductal adenocarcinoma [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 1561.