bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 2868: ADG206, an anti-CD137 agonistic POWERbody࣪ with tailor-made efficacy and safety profiles by strong crosslinking and tumor selective activation for single and combinational cancer immunotherapy
Authors: Liu Guizhong , Dai Zhengxi , Qu Jiagui , Shi Jianfeng , Zhao Qi , Qu Linhai , Li Yan , Zheng Songmao , Xu Jiangchun , Du Felix , Luo Peter
Published: June 2022

Targeting CD137 by agonistic antibodies faces various challenges in the clinic. Utomilumab appears safe but has modest efficacy, while urelumab is more efficacious but causes severe dose-dependent liver toxicity. To unlock the full potential of CD137 immunotherapy, we engineered a ADG206 POWERbody࣪ by combining our SAFEbody® technology’s precision masking of the antigen binding sites with the Fc engineering in order to achieve improved safety and efficacy profiles. In normal tissues, the CD137 antigen binding interfaces of ADG206 are masked using a covalently linked designer peptide to limit on-target off-tumor toxicities, whereas in the tumor microenvironment (TME), the masked antibody can be activated to selectively bind CD137, promoting costimulatory signaling in situ. The activity of ADG206 is further heightened by incorporating Fc mutations with increased FcγR-mediated crosslinking, resulting in enhanced T cell responses and antitumor activity. In vitro nonclinical studies demonstrate that activated ADG206 binds to CD137, with agonistic activity strictly dependent on FcγR-mediated crosslinking, in contrast to FcγR-independent stimulation of CD137 by urelumab. When activated, ADG206 exhibits up to ~4-fold stronger anti-CD137 agonistic activity than urelumab for T cell activation in the presence of a primary stimulatory signal, while masked ADG206 has much lower activity. In vivo nonclinical studies demonstrate that ADG206 has robust single agent antitumor activities in multiple syngeneic murine tumor models and synergistic efficacy when combined with other immunomodulatory agents. Moreover, ADG206 is well-tolerated in rats and cynomolgus monkeys in nonclinical toxicology studies, with normal pharmacokinetic behaviors and minimal activation in circulation. These results demonstrate that ADG206 POWERbody acquires a tailor-made product profile toward a best-in-class anti-CD137 immunotherapeutic agent that combines conditional activation in the TME with strong agonistic activity through heightened FcγR-mediated crosslinking. Further clinical development of ADG206 POWERbody may hold promises for achieving the long-awaited desirable tolerability and efficacy outcome of an anti-CD137 immunotherapy in either single agent or combination regimens.

Citation Format: Guizhong Liu, Zhengxi Dai, Jiagui Qu, Jianfeng Shi, Qi Zhao, Linhai Qu, Yan Li, Songmao Zheng, Jiangchun Xu, Felix Du, Peter Luo. ADG206, an anti-CD137 agonistic POWERbody࣪ with tailor-made efficacy and safety profiles by strong crosslinking and tumor selective activation for single and combinational cancer immunotherapy [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 2868.
Abstract 2905: A novel therapeutic antibody against CD73 that ameliorates the tumor microenvironment and improves the efficacy of cancer immunotherapy
Authors: Matsumoto Noriko , Mishima Yuji , Obonai Toshifumi , Ban Haruka , Mie Motoya , Nakamura Norihiro
Published: June 2022

Background: CD73, also known as ecto-5’-nucleotidase (NT5E), is a GPI-anchored cell surface enzyme expressed on certain types of immune cells and tumor cells. This enzyme catalyzes the hydrolytic conversion of extracellular adenosine monophosphate (AMP) to adenosine (Ado) that suppresses tumor-infiltrating immune cells via the adenosine receptors, allowing tumors to escape the immune system. Here, we introduce a novel anti-human CD73 antibody called BP1200 that strongly blocks the generation of Ado by CD73 and thereby reverses immune suppression in tumors.

Methods: We screened anti-CD73 antibodies by antigen-specific single B cell sorting from mice immunized with recombinant human CD73. CDRs of the antibodies that inhibit the enzyme activity of CD73 were engrafted into human IgG1 framework carrying the mutations of Fc silent. The proliferation and cytotoxicity of T cells were analyzed using human peripheral blood mononuclear cells (PBMC) in the presence of ATP. Efficacy of BP1200 in vivo was investigated by using mice bearing human CD73 knock-in-MC38 cells and mice implanted with human NSCLC cell line (NCI-H292) and PBMC.

Results: The humanized anti-CD73 antibody (BP1200) binds to both human and cynomolgus CD73 expressed on cell surface with KD value of 0.2 nM (human CD73). BP1200 blocked the hydrolysis of AMP by CD73 expressed as both membrane-bound and soluble form without the hook effect. BP1200 induced internalization of CD73 and remarkably decreased the cell surface CD73, indicating that BP1200 inhibits the enzyme activity as well as downregulates the protein level of CD73 at the cell surface. BP1200 induced in vitro proliferation, cytokine production, and cytotoxic activity of T cells even in the presence of high level of ATP, suggesting that BP1200 enhances the T cell immunity in immunosuppressive tumor micro-envelopment (TME). In vivo studies showed that the combination treatment of BP1200 with anti-PD-1/PD-L1 antibodies remarkably retarded tumor growth. Moreover, the mice administrated with BP1200 and anti-PD-1 exhibited long-term immune memory effects that rendered them resistant to rechallenge with tumors.

Conclusions: The novel anti-human CD73 antibody, BP1200, ameliorates immunosuppressive TME caused by adenosine and enhances antitumor immune responses. The combination of BP1200 and immune-checkpoint inhibitors for cancer treatment will be a promising therapeutic option in clinical practice.

Citation Format: Noriko Matsumoto, Yuji Mishima, Toshifumi Obonai, Haruka Ban, Motoya Mie, Norihiro Nakamura. A novel therapeutic antibody against CD73 that ameliorates the tumor microenvironment and improves the efficacy of cancer immunotherapy [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 2905.
Abstract 2944: Rapid and accurate variant calling of FFPE samples with the Genexus System
Authors: Casuga Iris , Chan Frances , Huynh Milton , Govoni Gregory R. , Zochowski Kayla , Jayaweera Thilanka , Au-Young Janice
Published: June 2022

Next-Generation Sequencing technology has enhanced oncology research by enabling the detection of all cancer related variants into one assay for research and drug discovery programs. The Oncomine࣪ Comprehensive Assay v3, a pan-cancer panel, used with the Ion Torrent࣪ Genexus࣪ System allows for formalin-fixed paraffin embedded (FFPE) samples to be examined across 161 unique genes in an automated sample to result workflow in 30hrs. This study demonstrates ≥ 95% Sensitivity and PPV for detecting SNV, Indel, and Copy Number variants of clinical FFPE samples along with fusions. When tested using analytical controls, the Genexus System achieves ≥ 98% Sensitivity for hotspot variants, ≥ 95% for de novo variants, and 100% Sensitivity for fusion variants. The Ion Torrent࣪ Genexus࣪ System is fully automated and consists of two software linked instruments, the Ion Torrent࣪ Genexus࣪ Purification System and the Ion Torrent࣪ Genexus࣪ Integrated Sequencer. For this study, the purification system was used to sequentially extract and quantify DNA & RNA samples from human colon and lung FFPE tumor tissue. The purification instrument provides minimal hands-on-time, ease of use and compatibility with the integrated sequencer. It extracts and quantifies the nucleic acids, records the quantitation values and transfers the nucleic acids to an output plate. The plate is transferred from the purification instrument directly to the sequencer for sample dilution, library preparation, and sequencing using the Oncomine࣪ Comprehensive Assay v3 panel. Variant calling analysis of the DNA & RNA pairs is completed immediately following sequencing. Results show that the variants are detected reproducibly with ≥ 95% Sensitivity and PPV when compared to the Ion GeneStudio࣪ S5 System as an orthogonal method. All variants in the report were correct with p-values ≤ 10-5. Additionally, a subset of the extracted samples was evaluated using Sanger Sequencing to verify hotspot mutations found in samples sequenced on both the Genexus࣪ System and GeneStudio࣪ S5 System. Results show that all expected hotspot variants were detected. In summary, the Ion Torrent࣪ Genexus࣪ System is a reliable and fast turnaround solution for sample-to-variant calling results. When used with the Oncomine࣪ Comprehensive Assay v3 panel, the system provides accurate identification of tumor markers for oncology research.

Citation Format: Iris Casuga, Frances Chan, Milton Huynh, Gregory R. Govoni, Kayla Zochowski, Thilanka Jayaweera, Janice Au-Young. Rapid and accurate variant calling of FFPE samples with the Genexus System [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 2944.
Abstract 312: Redirecting T cells to tumor targets with functionally diverse CD3-binding antibodies
Authors: DeVorkin Lindsay , Jacobs Tim M. , Tonikian Raffi , Hervé Karine , Caldwell Kate , Hwang Yuri , Faralla Cristina , Wei Wei , Lam Katherine J. , Dhupar Harveer , Tran Tran NT , Cid Melissa , Bolten Lena M. , Pinsky Tova , Dalal Kush , Heyries Kevin A. , Barnhart Bryan C.
Published: June 2022

Bispecific antibodies that redirect cancer-killing T cells towards tumors are promising next-generation cancer therapies. While there are hundreds of T cell engagers (TCEs) in development, there is only one approved and marketed CD3-binding TCE. The high rate of attrition is largely attributable to dose-limiting toxicities, including cytokine release syndrome, due in part to the small pool of high affinity CD3-binding antibodies that are commonly used. The discovery of safe and effective TCEs is limited because diverse panels of parental CD3 antibodies are hard to produce, the pairing of parentals is hard to perfect, and the sheer complexity and volume of data is hard to action. In this study, we will present a panel of functionally diverse, fully human CD3-binding parental antibodies. We will present data characterizing the diversity of our panel across multiple parameters, including sequence diversity, CD3 affinity, epitope binding, T cell activation, cytokine release, and tumor cell killing. Using OrthoMab࣪, our clinically-validated bispecific engineering platform, allows this panel to be tested with a series of tumor-associated antigens (TAAs) in a matrix format. Results from high-throughput production and characterization of bispecific antibodies will be presented. These multidimensional datasets of TCE composition and function allow for the identification of pairs that are optimal candidates for clinical development. This work will show how the diversity of our CD3-binding panel, combined with a robust bispecific protein engineering technology, can be used to quickly assess large and diverse TAA-binding panels discovered through our technology stack. An integrated workflow that doubles the data with diverse panels of parentals, assembles stable, safe, and manufacturable TCEs, and visualizes multidimensional datasets are critical to successfully identifying lead therapeutic candidates to bring the next generation of cancer therapies to patients sooner.

Citation Format: Lindsay DeVorkin, Tim M. Jacobs, Raffi Tonikian, Karine Hervé, Kate Caldwell, Yuri Hwang, Cristina Faralla, Wei Wei, Katherine J. Lam, Harveer Dhupar, Tran NT Tran, Melissa Cid, Lena M. Bolten, Tova Pinsky, Kush Dalal, Kevin A. Heyries, Bryan C. Barnhart. Redirecting T cells to tumor targets with functionally diverse CD3-binding antibodies [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 312.
Abstract 3170: Human retroviral sequences transcription in glioma/glioblastoma sample
Authors: Lassak Adam J. , Rak Monika , Baddoo Melody C. , Lin Hui-Yi , Reiss Krzysztof
Published: June 2022

In glioblastoma patients, expansion of glioma initiating cells (GICs), a discrete population of cells demonstrating stem-like phenotype, is often associated with drug resistance development and ultimately cancer recurrence. Therefore a better understanding of the molecular and cellular mechanisms underlying the origin of GICs is critical to develop new therapies better targeting glioblastoma. Human endogenous retroviral sequences (HERVs), present in approximately 500,000 copies and comprising nearly 8% of the human genome, may serve as an abundant genetic material equipping GIC with phenotypic plasticity needed to develop stem-like phenotype. Recent epigenomic and transcriptomic data show HERV activation during early human development. Moreover, increased HERV transcription is observed in OCT4/SOX2/KLF4/c-MYC induced pluripotent cells. Finally, activation of HERV loci transcription is also commonly observed in different tumor types. Using publicly available The Cancer Genome Atlas Glioblastoma Multiforme (TCGA-GBM) RNA-seq data collection, we have quantified transcripts for all 13,208 HERV loci annotated in the RepBase database. To improve the accuracy of quantifying the HERV transcriptomic landscape, we reassigned already mapped fragments to the most probable transcript using the Telescope software. Our comprehensive approach allowed us to identify the set of HERV loci uniquely dysregulated in the glioblastoma compared to normal brain tissue. In addition, we compared 540 low-grade glial tumors from TCGA-LGG project (classified as grade II and III gliomas) with 165 glioblastoma samples from TCGA-GBM (classified as grade IV gliomas) using the same bioinformatics pipeline. Comprehensive analysis of transcript levels allowed us to identify HERV assemblage differentially expressed between these two patient cohorts. The correlation between HERV and ORF transcript levels show strong coregulation of HERVs expression with genes involved in establishing/maintaining embryonic/neural/glial stem cells phenotype. To confirm our observations we have up- or downregulated HERV loci combining guide RNA array targeting robustly approximately 90% of HERV LTR5Hs elements with a vector expressing CRISPR-based transcriptional activator or repressor. Downregulation of HERV transcription with this system was associated with the inhibition of clonogenic growth in cell culture. In summary, this study sets the stage for identifying HERV loci that could serve as potential GIC biomarkers, as well as the molecular targets for GIC elimination.

Citation Format: Adam J. Lassak, Monika Rak, Melody C. Baddoo, Hui-Yi Lin, Krzysztof Reiss. Human retroviral sequences transcription in glioma/glioblastoma sample [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 3170.
Abstract 3175: Methylmalonic acid in TME signaling
Authors: Low Vivien , Li Zhongchi , Laughney Ashley , Jin Wenbing , Dephoure Noah , Luga Valbona , Earlie Ethan , Parang Bobak , Guo Chunjun , Cantley Lewis , Blenis John
Published: June 2022

The importance of metabolic reprogramming in cancer has been long demonstrated by the association of systemic metabolic changes, such as aging, diet and exercise, with cancer outcomes. These systemic shifts, combined with local metabolic alterations within the tumor microenvironment (TME), can all cooperate to foster an environment conducive to cancer progression. In cells derived from primary and metastatic patient tumors, we found that mesenchymal-like cells displayed dysregulated propionate metabolism, leading to increased accumulation and secretion of methylmalonic acid (MMA), a novel aging-induced oncometabolite. This tumor cell-secreted MMA, in addition to increased MMA in the serum of elderly individuals, combine to form high local accumulation of MMA in the TME. We discovered that MMA acts on fibroblasts in the TME, activating them to cancer-associated fibroblasts (CAFs). MMA modifies the cargo of CAF-secreted extracellular vesicles (EVs), which function as a messenger to tumor cells, further promoting epithelial-to-mesenchymal transition, drug resistance, and increased metastasis in vivo. Here, we reveal a novel function of MMA in cancer, demonstrating for the first time that tumor-secreted MMA recruits the tumor microenvironment to drive cancer progression and metastatic reprogramming.

Citation Format: Vivien Low, Zhongchi Li, Ashley Laughney, Wenbing Jin, Noah Dephoure, Valbona Luga, Ethan Earlie, Bobak Parang, Chunjun Guo, Lewis Cantley, John Blenis. Methylmalonic acid in TME signaling [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 3175.
Abstract 3177: Liver endothelium secreted LRG1 is a novel ligand of HER3 to promote metastatic colorectal cancer growth
Authors: Rathore Moeez Ghani , Zhang Wei , Wright Michel'le , Winter Jordan , Li Yamu , Wang Zhenghe , Wang Rui
Published: June 2022

Background: Liver is the most common site of developing distant metastasis in colorectal cancer (CRC). Patients with metastatic CRC (mCRC) have a 5-year survival rate at 14%. We previously showed that liver endothelial cells (EC), a key component of the liver microenvironment, secrete soluble factors to promote CRC growth and chemoresistance via activating human epidermal growth factor receptor (ERBB3, also known as HER3). However, we found that the ECs activated HER3 by a previously unknown mechanism that is independent of the established HER3 ligand neuregulins. Identifying a new HER3 ligand will potentially give rise to novel therapeutic target for treating patients with mCRC.

Methods: We first fractionated conditioned medium (CM) from liver ECs by fast protein liquid chromatography. The specific fractions that activated HER3 and AKT in CRC cells were subjected to mass spectrometry (MS) for protein identification. In parallel, we used a His-tagged HER3 Extracellular domain (ECD) and pulled down EC-secreted factors that directly bound to HER3 and then subjected to protein identification by MS. Candidate proteins identified in both MS analyses were subjected for further validations by siRNA or immunoprecipitation depletion from EC CM to determine their roles in HER3 activation and CRC growth.

Results: We identified a specific fraction of liver EC CM increased phosphorylation of HER3 and AKT in CRC cells (determined by Western blotting). Meanwhile, the HER3-ECD affinity binding assay showed that HER3 ECD-depleted EC CM could no longer activate HER3 or promote growth, and the soluble factor(s) eluted from HER3-ECD increased phosphorylation of HER3 and AKT, and promoted growth in CRC cells. Results from both MS analyses revealed that LRG1 is a potential ligand that binds to and activate HER3 in CRC cells. We depleted LRG1 from EC CM, either by siRNA knockdown LRG1 in ECs or immunoprecipitation, and found that LRG1-depleted EC CM could no longer activate HER3 and AKT in CRC cells, and LRG1-depleted EC CM failed to promote growth of CRC cell in vitro and CRC xenografts in vivo.

Conclusions: We identified LRG1 as a new HER3 ligand and demonstrated that liver EC-secreted LRG1 mediates EC paracrine effects on activating HER3 and promoting CRC cell growth. Our findings suggest an oncogenic role of EC-secreted LRG1 in the surrounding liver microenvironment of mCRC, and highlighted a potential of blocking LRG1 with monoclonal antibodies for treating patients with mCRC.

Citation Format: Moeez Ghani Rathore, Wei Zhang, Michel'le Wright, Jordan Winter, Yamu Li, Zhenghe Wang, Rui Wang. Liver endothelium secreted LRG1 is a novel ligand of HER3 to promote metastatic colorectal cancer growth [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 3177.
Abstract 3192: EBV-products containing exosomes remodel NPC tumor microenvironment by activating fibroblasts
Authors: Lee Po-Ju , Sui Yun-Hua , Huang Chen-Han , Liu Tzu-Tung , Tsang Ngan-Ming , Liu Shu-Chen
Published: June 2022

Epstein-Barr virus (EBV) infection is intimately associated with type III nasopharyngeal carcinoma (NPC). Most NPCs respond well to chemoradiation. Nevertheless, approximately 20% of patients experience recurrence and distant metastasis. Tumor desmoplastic responses and high densities of cancer-associated fibroblasts (CAFs) within tumors have been correlated with poorer prognosis. In this study, we demonstrate that EBV activates fibroblasts via exosomes to remodel a pro-tumoral microenvironment. Exosomes derived from NPC cells contained a high level of fibroblast activation protein alpha (FAPα) and EBV-encoded latent membrane protein 1 (LMP1). Treating primary fibroblasts with these exosomes induced functional changes resembling CAFs. Transcriptomic analysis on exosome-treated fibroblasts revealed genes involved in fibrosis-associated pathways were enriched. Noticeably, treating with EBV products-containing exosomes in fibroblasts enhanced the production of several immune-suppressive cytokines, including IL6, IL8, and MCP-1. Moreover, stimulation with these exosomes enhanced activation of YAP1 signaling, a key pathway linking to fibrosis. The in vivo mouse xenografted NPC studies showed that EBV products-containing exosomes markedly enhanced the severity of tissue fibrosis and tumor growth. IHC analysis on mouse NPC xenografts and human NPC biopsies showed a positive correlation between levels of nuclear YAP1 and FAPα, which were further correlated with an enhanced fibrotic response within tumors. Pharmaceutical intervention with YAP1 inhibitors blocked the exosome-mediated effects on fibroblasts. Collectively, our data suggest that EBV products-containing exosome is a causal factor in CAF formation, which constitutes a pro-tumoral microenvironment to benefit the survival of both tumor and virus itself.

Citation Format: Po-Ju Lee, Yun-Hua Sui, Chen-Han Huang, Tzu-Tung Liu, Ngan-Ming Tsang, Shu-Chen Liu. EBV-products containing exosomes remodel NPC tumor microenvironment by activating fibroblasts [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 3192.
Abstract 3284: HB-201 and HB-202, an arenavirus-based immunotherapy, induces tumor T cell infiltration in patients with HNSCC and other HPV16+ tumors
Authors: Edwards Donna , Schwendinger Michael , Katchar Kia , Schlienger Katia , Orlinger Klaus , Matushansky Igor , Lauterbach Henning
Published: June 2022

Background: Human Papillomavirus 16 (HPV16) is the most common etiologic agent of HPV-associated cancers. Treatment options are limited for patients with HPV16+ recurrent or metastatic cancers. HPV16 E7 and E6 oncogenes constitute attractive immunotherapeutic targets. HB-201 and HB-202 are live-attenuated replicating vectors based on LCMV and PICV arenaviruses, respectively, that express the same non-oncogenic HPV16 E7E6 fusion protein for induction of tumor specific T cell responses.

Methods: A Phase I/II open labelled clinical trial of HB-201 single vector therapy and HB-202/HB-201 alternating two-vector therapy in patients with treatment-refractory HPV16+ cancers is currently ongoing (NCT04180215). To assess circulating E6/E7 specific T cell responses, IFN-γ enzyme-linked immunospot (ELISpot) and intracellular cytokine staining were performed on pre- and post-treatment peripheral blood mononuclear cells (PBMCs). Evaluation of T cell infiltration and PD-L1 status in tumor bed was performed on paired tissue biopsies from a subset of patients by multiplex immunofluorescence.

Results: Single vector therapy (HB-201) and alternating two-vector therapy (HB-202/HB-201) rapidly induce high E6/E7 specific T cell levels, reaching up to ~40% of the circulating CD8+ T cell pool. Alternating two-vector therapy seems to maintain E6/E7 specific T cell responses better in continuous dosing compared to single vector therapy. Furthermore, we demonstrate tumor tissue T cell infiltration in more than 50% of patient samples analyzed. Currently, in depth sequencing of paired biopsies is underway to characterize the tumor microenvironment in response to HB-201 and HB-202/HB-201 treatment.

Conclusion: In this updated dataset, we show that HB-201 and HB-202/HB-201, rapidly induce unprecedented E6/E7 specific T cell levels in circulation following a single dose. Furthermore, these data are seen in conjunction with a pronounced increase of post-treatment CD8+ T cells in tumor, suggesting E6/E7 specific T cell infiltration. Our arenavirus vectors expressing the E7E6 fusion antigen demonstrate an attractive and safe therapy for patients with treatment refractory HPV16+ cancers. The ability of replicating arenavirus vectors to incorporate a broad range of antigens and the potent T cell inducing capacity provide a strong rationale to apply this novel therapy to other cancers.

Citation Format: Donna Edwards, Michael Schwendinger, Kia Katchar, Katia Schlienger, Klaus Orlinger, Igor Matushansky, Henning Lauterbach. HB-201 and HB-202, an arenavirus-based immunotherapy, induces tumor T cell infiltration in patients with HNSCC and other HPV16+ 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 3284.
Abstract 3326: Uncovering the molecular mechanisms which predict sensitivity to a novel src kinase inhibitor NXP900 to inform personalized healthcare strategies
Authors: Carragher Neil , Hughes Rebecca , Brunton Valerie , Unciti-broceta Asier , Lanzagorta-Calvillo Iñigo , Temps Carolin , Poradosu Enrique
Published: June 2022

We have previously described the discovery of a highly potent, selective and orally bioavailable Src kinase inhibitor NXP900 with a unique binding mode relative to other Src inhibitors under clinical development or approved (Temps et al 2021). This unique binding mode locks SRC in its native inactive conformation, thereby inhibiting both enzymatic and scaffolding functions. Also, in contrast to other Src inhibitors in the clinic, NXP900 exhibits a unique target selectivity profile with 1000 fold selectivity for Src over Abl kinase. The aim of this current study was to perform broad cancer cell line panel screening to inform disease positioning and identify predictive biomarkers of sensitivity for this novel class of Src kinase inhibitor. We describe the results of extensive cancer cell line panel profiling across a suite of 2-dimensional(D) and 3-D in vitro cell viability and high content phenotypic profiling assays that has revealed clear patterns of cell line sensitivity and insensitivity. Among the most sensitive cell line subtypes are, squamous cell carcinoma, Arid1A mutant ovarian clear cell carcinoma and subtypes of breast luminal A and triple negative breast cancer cell lines. HER2+ breast cancer lines appear insensitive to NXP900 indicating HER2 as a potential driver of resistance to NXP900. Further in-depth bioinformatic analysis of genetic and transcriptomic profiles of sensitive cell lines has identified putative biomarkers to support patient selection and personalized healthcare strategies. These studies demonstrate the identification of cancer cell lines which exhibit high sensitivity to NXP900 and which can be grouped into discrete molecular subtypes based on gene mutation status, transcriptomics and histotype. Our result provide an indication of patient subgroups which may exhibit optimal therapeutic response to NXP900 and provide data to guide further preclinical, biomarker and clinical development. Temps C et al., Cancer Res. 2021 Nov 1;81(21):5438-5450. doi: 10.1158/0008-5472.CAN-21-0613.

Citation Format: Neil Carragher, Rebecca Hughes, Valerie Brunton, Asier Unciti-broceta, Iñigo Lanzagorta-Calvillo, Carolin Temps, Enrique Poradosu. Uncovering the molecular mechanisms which predict sensitivity to a novel src kinase inhibitor NXP900 to inform personalized healthcare strategies [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 3326.
Abstract 3510: SOT201 is a novel targeted IL-15Rbg agonist to alleviate PD-1-mediated immune cell suppression and potentiate anti-tumor efficacy
Authors: Adkins Irena , Antosova Zuzana , Danova Klara , Hladikova Kamila , Augustynkova Katerina , Sajnerova Katerina , Podzimkova Nada , Marasek Pavel , de Martynoff Guy , Bechard David , Moebius Ulrich , Spisek Radek
Published: June 2022

SOT201 is a novel immunocytokine consisting of a monoclonal humanized, Fc silenced antibody against PD-1 fused to a covalent RLI-15 complex of a human IL-15 mutein linked to the high-affinity binding site of the IL-15Rα, the sushi+ domain. SOT201 is developed for immunotherapeutic treatment of various types of cancers. The activity of SOT201 is based on spatiotemporal reinvigorating of anti-tumor immune responses by disrupting co-inhibitory T-cell signaling by blocking PD-1 and synergistically activating adaptive as well as innate immunity by IL-15-mediated signaling via the IL-2/IL-15βγ receptor on T cells, NK, NKT, and γδ T cells. SOT201 showed a superior potentiation of T cell stimulation over pembrolizumab in mixed lymphocyte reaction in vitro. Studies in cynomolgus monkeys showed that decreased affinity of IL-15 mutein in SOT201 for its IL-15Rβγ is well optimized to ensure favorable pharmacokinetic properties while inducing strong CD8+ T cell and NK cell activation and expansion. Synergistic action on CD8+ T cell activation of both anti-PD-1 and RLI-15 moieties was confirmed using mouse surrogate SOT201 molecules in vivo. Strong anti-tumor efficacy after SOT201 treatment was achieved in a human PD-1 transgenic mouse model implanted with the MC38-hPD-L1 cell line. These data represent a promising therapeutic candidate molecule leveraging the synergistic concerted action of anti-PD-1 blockage and simultaneous immune cell activation directed preferentially to the high PD-1+ T cell tumor environment. The therapeutic potential of SOT201 is currently being prepared for evaluation in a Phase I clinical study in metastatic advanced cancer patients as well as for PD-1 resistant/refractory patients as our clinical stage IL-2/IL-15Rβγ agonist SOT101 already showed a clinical benefit in these patients in its ongoing Phase I study.

Citation Format: Irena Adkins, Zuzana Antosova, Klara Danova, Kamila Hladikova, Katerina Augustynkova, Katerina Sajnerova, Nada Podzimkova, Pavel Marasek, Guy de Martynoff, David Bechard, Ulrich Moebius, Radek Spisek. SOT201 is a novel targeted IL-15Rbg agonist to alleviate PD-1-mediated immune cell suppression and potentiate anti-tumor efficacy [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 3510.
Abstract 357: Characterization of KRas pathway inhibitors in 2D and 3D screening formats
Authors: Schaich Katharina , Feger Daniel , Siedentopf Oliver , Ulrich Sarah , Ehlert Jan Erik
Published: June 2022

The KRas/Raf/MEK signalling axis has been identified to play a critical role in the formation of cancer, resulting in several investigational new drugs targeting this pathway. Identification of appropriate drugs is hampered, however, by the assumption that a three-dimensional setting may be required to observe significant inhibitory effects, possibly due to alternate gene expression and protein activity in such a “closer-to-physiology” setting. In order to explore that topic, in the current study we have made use of our 140 cell lines (CL)-2D proliferation panel (3 days incubation) and our 100CL-3D soft agar panel (> 7 days incubation). On these platforms, we have compared inhibitors targeting the KRas/Raf/MEK pathway via either KRas:SOS interaction (e.g. BI3406), the KRas mutation at G12C (e.g. AMG510), or Raf-kinase (e.g. AZ628) and MEK1/2-kinase activity (e.g. AZD6244). It showed that especially the Raf- and MEK kinase inhibitors exhibited significantly enhanced potency in the 3D soft agar setting in the majority of the cell lines tested. The other inhibitors also showed an increased potency under soft agar conditions, however, on clearly fewer cell lines and usually not as strongly. For all kinds of inhibitors tested, the most significant changes in potency were associated with enhanced efficacy as observed by stronger cell death induction. Differences may be attributable to three-dimensional growth, but also to the elongated duration of the soft agar assay. For selected conditions, we addressed this topic by analyzing the inhibitor impact on 3D spheroid growth after shorter incubation times. Our results show that 3D growth analysis either in the soft agar or spheroid setting clearly supports the development of KRas/Raf/MEK pathway inhibitors.

Citation Format: Katharina Schaich, Daniel Feger, Oliver Siedentopf, Sarah Ulrich, Jan Erik Ehlert. Characterization of KRas pathway inhibitors in 2D and 3D screening formats [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 357.
Abstract 3573: DNA immunotherapy targeting BARF1 demonstrates therapeutic impact in novel murine carcinoma models expressing EBV latent antigens
Authors: Zhu Xizhou , Perales-Puchalt Alfredo , Wojtak Krzysztof , Xu Ziyang , Yun Kun , Bhojnagarwala Pratik S. , Bordoloi Devivasha , Park Daniel H. , Liaw Kevin , Bah Mamadou A. , Lieberman Paul M. , Gary Ebony N. , Patel Ami , Weiner David B.
Published: June 2022

Epstein Barr Virus (EBV) latent infection is associated with multiple types of cancer. Several clinical studies have targeted EBV antigens as immune therapeutic targets with limited efficacy of EBV+ malignancies, suggesting additional targets might be important. BamHI-A rightward frame 1 (BARF1) is an EBV antigen that is highly expressed in EBV-associated gastric carcinoma (EBVaGC) and EBV+ nasopharyngeal carcinoma (NPC) and has not been evaluated as an antigen in the context of therapeutic immunization. Here, we developed a synthetic DNA-based expression cassette as immunotherapy targeting BARF1 (pBARF1). Immunization with pBARF1 induced potent antigen-specific humoral and polyfunctional effector T cell responses in vivo. We observed significant antigen-specific increases of IFN-γ+, IFN-γ+ TNF-α+, and IFN-γ+ TNF-α+ IL-2+ populations in CD8+ T cells from the pBARF1 immunized animals in both C57BL/6 and BALB/c mice. Novel BARF1 expressing carcinoma lines that grow in immune-competent animals were developed as surrogates for human EBV+ tumors. We confirmed that BARF1 expression in these new cancer cell lines, MC38-BARF1 and CT26-BARF1, was within the range of human EBV+ cancer cell lines, SNU719 and C666-1. Immunization with pBARF1 plasmid demonstrated impact of tumor progression through induction of CD8+ T cell in these BARF1+ carcinoma models. We also observed long-term antitumor immunity, at least up to 446 days, against BARF1 in a CT26-BARF1 rechallenge study. Using IVIS in vivo imaging, we observed that pBARF1 immunized animals rapidly controlled and cleared cancer cells. These studies demonstrate the potential for pBARF1 immunity to induce antigen-specific immune responses impacting tumor progression. Further study of this immune target and these EBV+ tumor models is likely important as part of therapeutic approaches for EBV+ malignancies.

Citation Format: Xizhou Zhu, Alfredo Perales-Puchalt, Krzysztof Wojtak, Ziyang Xu, Kun Yun, Pratik S. Bhojnagarwala, Devivasha Bordoloi, Daniel H. Park, Kevin Liaw, Mamadou A. Bah, Paul M. Lieberman, Ebony N. Gary, Ami Patel, David B. Weiner. DNA immunotherapy targeting BARF1 demonstrates therapeutic impact in novel murine carcinoma models expressing EBV latent antigens [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 3573.
Abstract 3583: Identifying and testing cancer-derived synthetic-lethal anti-SARS-CoV-2 targets
Authors: Pal Lipika R. , Cheng Kuoyuan , Nair Nishanth Ulhas , Martin-Sancho Laura , Sinha Sanju , Pu Yuan , Riva Laura , Yin Xin , Schischlik Fiorella , Lee Joo Sang , Chanda Sumit , Ruppin Eytan
Published: June 2022

Introduction: Despite the development of two mRNA vaccines, there is an urgent unmet need of finding new antiviral strategies. One such potential antiviral strategy is to target the synthetic lethal (SL) partners of transcriptionally altered genes in infected host cells, thereby selectively killing them to halt the infection at its heels (Mast FD, JCB, 2020).

Methods: Here we conduct a first proof-of-concept SL inference approach to predict anti-SARS-CoV-2 targets in a systematic genome-wide manner. This effort capitalizes on our recently published pipeline for inferring clinically relevant SL interactions in cancer (Lee et al, Cell, 2021). Based on the latter, we comprehensively analyzed multiple in vitro and in vivo bulk and single-cell RNA-sequencing datasets of SARS-CoV-2 infection to predict candidate antiviral targets that are SL with altered host genes. Importantly, as our predictions are fine-tuned based on the analysis of patients’ data, they are more likely to be of translational value.

Results: Our key results are twofold:1) The predicted SL-based targets are highly enriched for genes that are reported in four SARS-CoV-2 CRISPR-Cas9 genome-wide genetic screens to inhibit growth of infected cells. 2) A subset of top predicted 26 genes were experimentally tested in a targeted siRNA screen conducted in both infected and non-infected human Caco-2 cells. Remarkably, as expected given that these targets were predicted to be SL specific with genes upregulated in infected cells, indeed, knocking down these targets reduced viral replication and cell viability only under the infected condition without harming non-infected cells.

Conclusion: In summary, this study is the first to demonstrate the potential of a synthetic lethality approach to identify viral (specifically anti-SARS-CoV-2) targets. Importantly, as both single cell and bulk transcriptomics patients’ data is considered from both infected people and controls, they are more likely to be of clinical relevance. Targeting host genes identified via an SL-based approach is probably more suitable when the infection is at the early stage and host can still tolerate the loss of infected host cells.

Citation Format: Lipika R. Pal, Kuoyuan Cheng, Nishanth Ulhas Nair, Laura Martin-Sancho, Sanju Sinha, Yuan Pu, Laura Riva, Xin Yin, Fiorella Schischlik, Joo Sang Lee, Sumit Chanda, Eytan Ruppin. Identifying and testing cancer-derived synthetic-lethal anti-SARS-CoV-2 targets [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 3583.
Abstract 3607: A novel adjunctive LAT-activating CAR T (ALA-CART) cell platform demonstrates enhanced antigen sensitivity and eradication of antigen-low leukemia
Authors: Pham-Danis Catherine , Leach Lillie , Ebmeier Christopher , Kohler M. Eric
Published: June 2022

CAR T cells have demonstrated clinical success in malignancies that are refractory to standard therapies; however, preclinical and clinical studies have demonstrated that CAR T cell efficacy is greatly reduced in settings of low levels of target antigen. This was exemplified in a clinical trial of CD22-directed CAR T cells where despite inducing complete remissions in >70% of patients, post-CAR relapses occurred in the majority of patients, driven by decreased expression of CD22 below the threshold required for adequate CAR T cell activation. To address this limitation of CAR T cells, we investigated the mechanism underlying suboptimal CAR responses to low levels of antigen. Using a SILAC/mass spectrometry approach, we performed global phosphoproteomics to examine CAR T cell signal transduction in response to high- and low-levels of CD22 antigen. Low levels of CD22 antigen resulted in decreased utilization of several canonical T cell signaling pathways in CAR T cells. We hypothesized that LAT, a scaffolding protein integral to the T cell signalosome, was inefficiently utilized in response to low levels of antigen. Indeed, while signaling events proximal to LAT, such as Zap70, were unaffected by CD22 antigen density, phosphorylation of LAT at multiple activating residues decreased in response to stimulation with CD22-low leukemia cells. To overcome the inefficient LAT activation by low levels of antigen, we designed a bicistronic construct consisting of a clinically active 2nd generation CD22-BBz CAR along with a novel Adjunctive LAT Activating CAR incorporating the intracellular signaling domain of LAT (ALA-CART). ALA-CART cells expressed both CARs on the surface and demonstrated enhanced phosphorylation of LAT and PLCγ in response to CD22-low leukemia cells. In xenograft models, ALA-CART cells completely eradicated CD22-low leukemia, significantly extending survival of mice in comparison to the standard CD22-BBz CAR which only modestly slowed leukemia progression. Additionally, in mice engrafted with leukemia expressing WT-levels of CD22, we found the persistence of ALA-CART cell was significantly enhanced relative to standard CD22-BBz CAR T cells. Thus, by identifying deficits in CAR signaling, we rationally designed the ALA-CART platform to target antigen-low cells, overcoming a mechanism of resistance to standard CAR therapies with the potential to improve outcomes of patients receiving CAR therapies.

Citation Format: Catherine Pham-Danis, Lillie Leach, Christopher Ebmeier, M. Eric Kohler. A novel adjunctive LAT-activating CAR T (ALA-CART) cell platform demonstrates enhanced antigen sensitivity and eradication of antigen-low leukemia [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 3607.
Abstract 3618: Loss of SETD2 sensitizes kidney cancer cells to DNA methylation inhibitors by inducing viral mimicry and RNA mis-splicing
Authors: Li Hong-Tao , Jang Hyo Sik , Rohena-Rivera Krizia , Gujar Hemant , Liu Minmin , Kulchycki Justin , Zhou Xinyi , Zhao Shuqing , Jones Peter , Bhowmick Neil , Liang Gangning
Published: June 2022

Large-scale sequencing efforts of human cancers have identified recurrent mutations and deletions in a variety of chromatin-regulating proteins that modulate DNA methylation, histone modifications, and nucleosome positioning. The dysregulation of histone H3 lysine 36 (H3K36) methyltransferase, SETD2, through transcriptional or genetic aberrations is associated with worse clinical outcomes and metastasis in kidney cancer. Limited targeted therapeutic interventions are available for aggressive SETD2-mutant ccRCC tumors. Here, we reveal that kidney cancer cells displaying diminished H3K36 trimethylation levels, a consequence of SETD2 deficiency, show increased sensitivity of anti-tumor effects to DNA hypomethylating agent (HMA). We discovered that HMA treatment induced stronger viral mimicry activation and immune upregulation, which is potentiated by higher transposable element (TE) expression in SETD2-mutant cancer cells. Mechanistically, we provide evidence that substantial number of the HMA-induced TE expression is a consequence of mis-splicing, which is associated deficient in slicing in SETD2-loss content along with rapid gain of H3K9me3 across exons. These all suggested HMA could turn immune-cold tumor to immune-hot tumor and sensitize tumors to immune therapy. Then we performed in vivo assay in immune competent mice. Indeed, SETD2 deficient tumors were extremely sensitive to combination treatment of HMA and immune checkpoint inhibitor. Our finding provides one of the first preclinical and in vivo evidence that demonstrates the SETD2 dysregulation can be an epigenetic therapeutic target in ccRCC, especially in combination with immune checkpoint inhibitors, for future clinical trials.

Citation Format: Hong-Tao Li, Hyo Sik Jang, Krizia Rohena-Rivera, Hemant Gujar, Minmin Liu, Justin Kulchycki, Xinyi Zhou, Shuqing Zhao, Peter Jones, Neil Bhowmick, Gangning Liang. Loss of SETD2 sensitizes kidney cancer cells to DNA methylation inhibitors by inducing viral mimicry and RNA mis-splicing [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 3618.
Abstract 3623: Targetable cofactor landscape of transcription factors in human cancers
Authors: Thatikonda Venu , Lipp Jesse , Schlattl Andreas , Gerlach Daniel , Popa Alexandra
Published: June 2022

Transcription factors (TF) are proteins that bind to DNA to regulate target gene expression and play a crucial role in maintaining tumor initiating and maintenance programs. However, except for a small fraction, it is difficult to directly target TF proteins due to lack of defined pockets and inherent protein-DNA, protein-protein interactions. In many cases, TF activity is tuned via cofactors, which e.g. dimerize to bind DNA (MYC/MAX) or maintain active TF protein turnover (HIF1A/EGLN1, MYC/HDAC2). Identifying such targetable cofactors of TFs will open new opportunities to target oncogenic TFs in cancers. Genome-scale CRISPR knock-out screens have been proven to be a valuable resource for identifying targets and biomarkers in cancers. In this work, we developed a framework called CONTEXT (COfactor depeNdencies in high Transcription factor EXpression phenoType) utilizing functional genomic screens and matched TF expression data. In CONTEXT, we aim to identify cofactor dependencies associated with TF activities using 859 cell lines spanning 28 cancer types. We further utilized patient cancer gene expression data from the TCGA project, to prioritize clinically relevant TF-cofactor associations. In total, we identified 1,003 TF-cofactor associations which involve 196 and 503 unique TFs and cofactors, respectively. Our approach reliably identified previously known TF-cofactor associations e.g. HIF1A/EGLN1, HIF1A/VHL, TEAD1/JUN but also previously unknown associations such as TEAD1/RAC1 or TEAD1/PTK2. Our analysis and validations provide a framework for the identification of potential cofactors of TFs which are crucial for the development and progression of cancer. We anticipate the cofactor dependencies from this resource to guide early stage drug development and inform on potential combination therapies in resistant phenotypes.

Citation Format: Venu Thatikonda, Jesse Lipp, Andreas Schlattl, Daniel Gerlach, Alexandra Popa. Targetable cofactor landscape of transcription factors in human 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 3623.