bluuebunny/crossref_metadata_2025_split huggingface dataset:
Abstract 2831: High-throughput T cell receptor cloning allows optimization of anti-miHA T cell therapies
Authors: Rowe Alexander M. , Didiano Dominic , Wei Wenzhong , Ito Sawa , Shlomchik Mark , Shlomchik Warren 
Published: June 2022

Abstract
Adoptive T cell therapy with CD8+ T cells engineered to express T cell receptors (TCRs) that recognize hematopoietically-restricted minor histocompatibility antigens (miHAs) is an attractive approach for improving the efficacy of allogeneic hematopoietic stem cell transplantation. We developed a mouse model to determine how to optimally implement anti-miHA T cell therapy, focusing on the key characteristics of anti-miHA TCRs that correlate with in vivo efficacy. We immunized B6 mice to the Kb-restricted miHA H60 and used a high throughput approach to amplify TCR α/β chains from single CD8+ MHC Multimer-H60+ (TetH60+) cells. Based on β chain sequencing of 472 single T cells, a broad repertoire was engaged by H60-vaccination, without a clear β chain preference. We randomly chose 178 unique TCRs, cloned them into retroviral backbones and expressed them in a TCR-CD8+ NFAT-reporter reporter cell lines and screened for H60-reactivity by NFAT-induction after stimulation with H60-pulsed antigen presenting cells (APCs). We chose 22 unique TCRs that had clear H60-specific NFAT-induction for deeper characterization. In similarly-transduced 4G4 reporter cells, these TCRs spanned a range of surface expression levels, affinities for H60 as measured by TetH60 binding, and EC50s in response to H60-pulsed APCs (between 1 and 12 μM peptide concentration). We focused initial functional studies on 2 high avidity TCRs (TCR 61 and TCR 167) with EC50s of 1.25 and 2.5 µM, respectively. These were cloned into an optimized retroviral vector and transduced into B6 CD8 cells concomitant with efficient CRISPR-knock out of the endogenous TCRα/β genes. Engineered T cells bound H60 loaded MHC multimers and killed H60+ targets in vitro. The mean TCR expression of transduced cells was 16-30% of that in unmanipulated T cells, though some had TCR expression similar to or greater than that in unmanipulated T cells. We used TCR 61 and 167 transduced T cell products to treat H60+ blast crisis leukemia cells (BC-CML). TCR 61 and 167 T cell products expanded and were equally effective at reducing BC-CML numbers in spleen and bone marrow (BM). Despite having a greater EC50 and equivalent surface expression in reporter cells, when recovered post-transplant, TCR167 cells had higher TetH60 binding, and produced more IFN-γ. Ongoing studies are testing these and additional TCRs alone and in competition to better elucidate TCR properties that predict for in vivo efficacy.
Citation Format: Alexander M. Rowe, Dominic Didiano, Wenzhong Wei, Sawa Ito, Mark Shlomchik, Warren Shlomchik. High-throughput T cell receptor cloning allows optimization of anti-miHA T cell therapies [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 2831.
Abstract 284: A 3D multicellular in vitro prostate cancer model featuring racially/ethnically diverse PDXs
Authors: Iyer Divya , Bonteanu Andrei , Shepherd Peter , Kittles Rick , Navone Nora , Harrington Daniel , Bircsak Kristin 
Published: June 2022

Abstract
Current in vitro prostate cancer (PCa) research tools do not incorporate the complexities of the tumor microenvironment including perfusion, multiple cell types, extracellular matrix (ECM) and 3D-orientation. Additionally, these models lack racial/ethnic (R/E) diversity, failing to address the cancer health disparity (CHD) observed in Black men with PCa who experience double the incidence and mortality rates as compared to White men. Population-based in vitro PCa models that support crosstalk between different cell types will improve our understanding of the underlying mechanisms contributing to this CHD and improve the prediction of drug response in specific populations. Here, we used the high-throughput, perfusion-based microfluidic platform called the MIMETAS OrganoPlate® to culture up to 96 individual, multicellular PCa-on-a-chip cultures in parallel. PCa PDX cells of various R/E were embedded together with E2Crimson-labeled stromal fibroblasts in a migration-permissive hyaluronic acid hydrogel in the gel compartment, alongside an endothelium-lined perfusion channel containing a targeted immune cell population. Using high content imaging, we confirmed the stability and viability of the cultures over 7 days. The cultures maintained a 3D structure with PCa cells and fibroblasts evenly dispersed throughout the height of the gel compartment and a blood vessel-like structure within the perfusion channel. Closer evaluation of the gel compartment revealed a close association between PCa cells and stromal fibroblasts in a core-shell structure with PCa clusters surrounded by fibroblasts. Further, all cells maintained expected expression of phenotypic markers (PCa: epCAM, PSMA; fibroblast: vimentin, endothelium: CD31). For the immune component, medium and cell tracker dye conditions were optimized to support immune cell viability. Over 72 hours, immune cells were monitored, revealing migration of PBMCs through the gel compartment. Finally, the value of the complex, population-based multicellular in vitro model of PCa will be evaluated in a drug screen comparing the system with simple, PCa PDX monocultures. This R/E diverse 3D prostate tumor model will enable the full incorporation of all cell types and ECM into a single model which will better recapitulate population specific PCa and drug response.
Citation Format: Divya Iyer, Andrei Bonteanu, Peter Shepherd, Rick Kittles, Nora Navone, Daniel Harrington, Kristin Bircsak. A 3D multicellular in vitro prostate cancer model featuring racially/ethnically diverse PDXs [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 284.
Abstract 2880: CD133-targeting silicate prodrug nanoparticles for effective cancer stem cell targeting
Authors: Bhatnagar Shubhmita , Jin Mengyuan , Hoye Thomas , Panyam Jayanth 
Published: June 2022

Abstract
Cancer stem cells (CSCs) are considered to be key contributors to drug resistance and tumor recurrence in many solid tumors. Therapeutic approaches that specifically target these cells are needed to improve treatment outcomes in these tumor types. We are investigating CD133, a widely used surface marker to identify CSCs in colon, brain, and breast tumors. Here, we report the use of polymeric nanoparticles targeting CD133 by conjugating an anti-CD133 scFv-Fc to poly(L-lactide)-PEG-maleimide nanoparticles (anti-CD133 NPs) for effective CSC targeting. The Fc tagged anti-CD133 scFv was prepared and purified using an Expi293 expression system. Nanoparticles were prepared using flash nanoprecipitation and loaded with a hydrophobic, hydrolytically labile, silicate prodrug of paclitaxel. Flash nanoprecipitation allowed for generation of particles with a size of 232 ± 17 nm (increased to 289 ± 23 nm upon scFv conjugation), a zeta potential of -11.5 mV and a drug loading of 57 ± 8 wt% measured by HPLC. The anti-CD133 scFv-Fc was thiolated using 2-iminothiolane (2-IT) and conjugated to nanoparticles using thiol-maleimide chemistry. Thiolation of the antibody was optimized using different molar excesses of 2-IT. A binding assay using Caco-2 cells (which overexpress CD133) confirmed that thiolation of the antibody did not affect its binding, even at the highest molar excess of 2-IT used. With 200x molar excess of 2-IT, 2.9 thiols per antibody were added. Antibody conjugation was confirmed using gel electrophoresis; bands corresponding to the heavy and light chains of antibody were observed for anti-CD133 NPs after reduction but were not seen for non-reduced anti-CD133 NPs or for unconjugated particles. Cytotoxicity studies in MDA-MB-231-LM2 cells revealed maximum kill in drug loaded anti-CD133 NPs, which was significantly higher than for either non-targeted, drug-loaded nanoparticles or blank nanoparticles (p&lt;0.05). Overall, these studies indicate the potential for using anti-CD133 scFv-Fc in conjunction with FNP-produced silicate prodrug nanoparticles of paclitaxel for efficient targeting of CSCs.
Citation Format: Shubhmita Bhatnagar, Mengyuan Jin, Thomas Hoye, Jayanth Panyam. CD133-targeting silicate prodrug nanoparticles for effective cancer stem cell targeting [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 2880.
Abstract 289: A novel ex-vivo organotypic culture platform for functional interrogation of human appendiceal neoplasms
Authors: Weitz Jonathan , de Mendoza Tatiana Hurtado , Tiriac Herve , Baumgartner Joel , Kelly Kaitlyn , Veerapong Jula , Lowy Andrew 
Published: June 2022

Abstract
Appendiceal neoplasms are rare and often clinically present with peritoneal metastasis. While surgical tumor resection is effective for some types of primary appendiceal cancers, patients with metastatic disease have poor prognostic outcomes. Models to study appendix cancer biology are limited, given that 1) no mouse models exist and 2) reliable in vitro models are unavailable. As such, we have developed an ex-vivo organotypic slice model to examine cellular interactions between tumor cells and their local microenvironment. Tumor specimens from human appendiceal cancer patients were cut using a vibratome to make 200 μm organotypic slices. Slices were cultured on transwell inserts and tested for changes in morphological, cellular and functional characteristics over a seven-day period. Organotypic slices maintained their cellular composition in regard to the proportion of epithelial, immune cells and fibroblasts. Live cell [Ca2+]i imaging of long term cultured slices confirmed that immune cells remain functionally active when stimulated with extracellular ATP. Lasty, using tumor biopsies from human donors, we have identified a diverse immunological profile of appendiceal tumors not previously identified. Our study illustrates a novel approach for studying the pathophysiology of appendiceal cancer, a notoriously difficult disease to model.
Citation Format: Jonathan Weitz, Tatiana Hurtado de Mendoza, Herve Tiriac, Joel Baumgartner, Kaitlyn Kelly, Jula Veerapong, Andrew Lowy. A novel ex-vivo organotypic culture platform for functional interrogation of human appendiceal neoplasms [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 289.
Abstract 2890: The development of an Adaptable Drug Affinity Conjugate (ADAC) targeting CD40 for a flexible therapeutic peptide cargo delivery to dendritic cells
Authors: Laurén Ida , Eltahir Mohamed , Mebrahtu Aman , Veerman Rosanne , Astorga Juan , Saleh Aljona , Ytterberg Jimmy , Lindqvist Annika , Dahllund Leif , Olsson Anders , Andersson Oskar , Rockberg Johan , Persson Helena , Mangsbo Sara 
Published: June 2022

Abstract
CD40 agonistic antibodies targeting antigen-presenting cells rely on simultaneous antigen presentation for optimal efficacy, as the co-stimulatory signal alone will not lead to T cell activation. Herein we have developed a novel Adaptable Drug Affinity Conjugate (ADAC), a refinement of a traditional Antibody Drug Conjugate (ADC) with a focus on tailored drug design. The cargo for ADAC is synthetic long peptides that can be modified with the profiled genetic data for each patient. The ADAC technology relies on a high-affinity interaction between a short non-immunogenic peptide-tag (pTag) and a single-chain fragment (scFv) and the targeting of CD40 expressing antigen-presenting cells. As a proof-of-concept study, we have generated data supporting a ~20 times improved half-life of peptide therapeutics bound up by the ADAC technology (in vitro), translating to improved peptide half-life in vivo along with a superior expansion of T cells in vitro and in vivo, compared to peptide and antibody mixed but not interacting physically. Through phage-display selection screening of novel CD40 agonistic antibodies, we identified a unique clone (STRIKE-1001). STRIKE-1001 binds a defined structural epitope (HDX-MS) of CD40 in a low nM range and is agonistic in an IgG2 format. Similar to selicrelumab, STRIKE-1001 does not block CD40L binding to Fc-CD40. STRIKE-1001 shows a dose-dependent agonistic activity (EC50 of ~28 nM based on measured induction of HLA-DR expression) on human DCs and can be designed into a bispecific antibody without loss of agonistic activity. STRIKE-1001 is currently incorporated into the ADAC platform to be used as the first-in-class candidate for flexible peptide cargo delivery ensuring T cell priming and expansion in vivo as a means to transform CD40 agonistic antibody to clinically relevant drug candidates.
Citation Format: Ida Laurén, Mohamed Eltahir, Aman Mebrahtu, Rosanne Veerman, Juan Astorga, Aljona Saleh, Jimmy Ytterberg, Annika Lindqvist, Leif Dahllund, Anders Olsson, Oskar Andersson, Johan Rockberg, Helena Persson, Sara Mangsbo. The development of an Adaptable Drug Affinity Conjugate (ADAC) targeting CD40 for a flexible therapeutic peptide cargo delivery to dendritic cells [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 2890.
Abstract 2893: Exploring the combinatorial potential of bispecific T-cell engagers in high throughput format
Authors: Castro Carla N. , Feger Daniel , Ulrich Sarah , Siedentopf Oliver , Ehlert Jan E. 
Published: June 2022

Abstract
Bispecific antibodies are being explored as a means to modulate responses of the immune system to tumor infiltration. They are emerging as a growing class of immunotherapies with potential to further improve clinical efficacy and safety. Besides tumor-targeted and dual immunomodulators, the most important class of therapeutically relevant bispecific antibodies are engagers of cytotoxic immune cells to kill tumor cells. These are mainly engager of T-cells (BiTE´s), but also NK-cell engager several of which have entered clinics with Blinatumomab (anti-CD3/anti-CD19) being FDA-approved for the treatment of Acute Lymphoblastic Leukemia. Several studies are under way to address potential combinatorial effects with other compounds in clinics. In order to facilitate analysis of the cytotoxic impact of BiTE´s in high throughput, we developed a 384-well format assay system based on Luciferase-labeled tumor cells. This detection technology has many advantages. As only the tumor cells are labeled, the specific detection of luciferase exclusively correlates to the number of viable tumor cells, allowing for co-cultures with high excess of effector cells without them interfering in the detection. Due to high sensitivity, only few tumor cells are required for a decent signal/noise ratio, keeping the overall need for primary effector cells to the lowest, even at high effector/target cell ratios. That can be of great advantage when working with rare cytotoxic subpopulations (e.g. NK-cells), precious samples from patients, or simply saves resources when performing large exploratory studies. In addition, combination of low 384-well format volumes with nanodrop agent dispensing technology minimizes amounts of expensive antibodies and other agents. Applying this technology, we evaluated the combinatorial effects of Blinatumomab with immune modulators (e.g. Lenalidomide), kinase inhibitors (e.g. MEK-inhibitor, Selumetinib) and other clinically relevant agents (e.g. MCL1-inhibitor, S63845) on the Luciferase-labeled GCB-like DLBCL cell line OCI-LY1. Depending on compound combination, we observe synergistic but also antagonistic effects. Our data support the outstanding usefulness of this methodological approach for the exploration of bispecific cytotoxic immune cell engager agents.
Citation Format: Carla N. Castro, Daniel Feger, Sarah Ulrich, Oliver Siedentopf, Jan E. Ehlert. Exploring the combinatorial potential of bispecific T-cell engagers in high throughput format [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 2893.
Abstract 2925: Expanding the library of small molecule imaging probes for heparanase
Authors: Schleyer Kelton , Liu Jun , Wang Zhishen , Guo Hua , Cui Lina 
Published: June 2022

Abstract
Heparanase is an enzyme highly involved in the progression of many types of cancer through its enzymatic processing of heparan sulfate proteoglycans. Unfortunately, research and clinical investigation of this target is hampered by the lack of simple, effective tools to detect its enzymatic activity. Despite the natural preference of heparanase to cleave polysaccharide substrates, we previously developed the first disaccharide-based fluorogenic probe for heparanase enzymatic activity, overcoming this deficiency in existing tools. However, the chemistry of the probe synthesis required installation of electron-withdrawing fluorine atoms on the fluorophore to facilitate activation by heparanase, a design strategy that excludes the use of many common imaging reporters. To accommodate a wider variety of imaging modalities for heparanase detection, we experimented with the addition of a collapsible linker with suitable electronegativity that can be easily tuned. Our results show that a tetra-fluorinated benzyl alcohol linker enables heparanase activation of a probe bearing any reporter, giving comparable results to our original fluorinated coumarin probe. This disaccharide-linker scaffold holds potential for accessing a broader scope of heparanase imaging probes for laboratory and clinical applications of this burgeoning biomarker.
Citation Format: Kelton Schleyer, Jun Liu, Zhishen Wang, Hua Guo, Lina Cui. Expanding the library of small molecule imaging probes for heparanase [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 2925.
Abstract 2932: The 6-color naica® system: a digital PCR platform and workflow for high multiplex genetic detection and analysis
Authors: Hoskins Andrew , Primus Shekerah , Yazdani Mary , Gutierrez Kimberley , Foret Benjamin , Bernard Fabien , Dangla Rémi 
Published: June 2022

Abstract
There are several molecular platforms routinely used to investigate genomic material. Quantitative polymerase chain reaction (qPCR) is ubiquitously used because of the ease of use and the precision however, it does not offer high multiplexing. While digital PCR (dPCR) is known for increased precision and sensitivity, most digital PCR platforms provide the same level of multiplexing as qPCR. Next-generation sequencing (NGS) is less precise than qPCR or dPCR and but has the highest multiplexing capability of the three technologies. Conventional digital PCR systems have been limited by the number of instruments required to conduct the workflow, time-to-result compared to qPCR, and level of multiplexing as compared to next-generation sequencing (NGS).
Crystal Digital PCR™ and the naica system is a next-generation digital PCR platform that simplifies the number of instruments in the workflow, speeds up the time of results to less than 3 hours, and increases the number of detection colors to 6 spectrally distinct channels. By using a combination of dual-labelled fluorescent probes, multiplexed digital PCR provides a simple solution that makes it possible to use less of a precious sample when faced with limited quantity without sacrificing sensitivity.
The naica system workflow utilizes microfluidic consumables, such as the Sapphire chip for high sensitivity applications, and the Opal chip for higher throughput studies. All reagents including primers and probes, master mix, and sample are inputted into the chips in one single loading step. Chips are transferred to the Geode instrument, where the sample is partitioned into a 2D-monolayer of droplet crystals and where all targets are amplified. After the PCR program has been completed, the same chips are then loaded into the Prism6 imager which detects all targets across 6 fluorescent channels and outputs an image of all droplets in a sample. Finally, Crystal Miner analysis software can be used to analyze and decipher the quantifications and expression of all targets in the assay.
The 6-color naica system allows for high multiplex detection of targets previously unattainable by conventional digital PCR systems. With this advent, genomic scientists have an additional platform in the molecular toolbox that supports seamless assay development of oncology biomarker assays for gene expression, rare target detection, and liquid biopsy applications.
Citation Format: Andrew Hoskins, Shekerah Primus, Mary Yazdani, Kimberley Gutierrez, Benjamin Foret, Fabien Bernard, Rémi Dangla. The 6-color naica® system: a digital PCR platform and workflow for high multiplex genetic detection and analysis [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 2932.
Abstract 295: Superior efficacy of targeted nanoparticle therapy for brain tumors
Authors: Kang HyungGyoo , Nagy Jon , Upton Bryon , Triche Timothy 
Published: June 2022

Abstract
Glioblastoma Multiforme is an incurable malignant brain tumor. Despite intensive efforts over many years, the prognosis is essentially unchanged. The primary reason is failure of otherwise effective therapy to reach the tumor, due to the blood brain barrier (BBB). To circumvent this functional barrier, we have developed CD99 and CD276 targeted irinotecan (Ir) containing liposomal nanoparticles (TNS: Targeted NanoSpheres) TNSs effectively penetrate the BBB and selectively bind to GBM cells while largely bypassing normal brain tissue. CD99 or CD276 targeted Ir-TNSs prolonged survival five-fold over free Ir treated animals in bi-weekly single dose level studies. Normal tissue levels were negligible, and no treatment-related toxicity was observed. QD dose intensification induced tumor regression in animals with progressive tumor. Anti-tumor effect was equivalent for either CD99 or CD276 Ir-TNSs. These results imply that intracranial therapy with otherwise effective drugs that do not pass the BBB is both feasible and shows superior efficacy over free drug therapy. Further, widespread expression of CD276 by many malignancies implies this therapy could be effective for metastatic brain tumors and extra-cranial malignancies as well.
Citation Format: HyungGyoo Kang, Jon Nagy, Bryon Upton, Timothy Triche. Superior efficacy of targeted nanoparticle therapy for brain tumors [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 295.