Established Tumors Research Articles

Abstract 3439: An internalizing anti-CEA construct for targeted delivery of granzyme B results in significant antitumor activity in human lung carcinoma xenografts

Abstract Carcinoembyronic antigen (CEA) is a cell surface protein normally found at low levels in adult tissues but over-expressed in numerous solid tumors. CEA is constitutively released from tumor cells and reaches detectable levels in peripheral blood, making CEA targeting particularly challenging. Granzyme B is a key effector in immune-mediated killing of target cells, with cell death through both caspase-dependent and -independent mechanisms. We developed a fusion protein composed of granzyme B and a human scFv targeting the juxta-membrane epitope of CEA. The construct also incorporates a fragment of human IgG heavy chain to cause dimerization and provide increased in vivo circulation and antitumor efficacy. The GrB-Fc-CEA construct was cloned into a mammalian expression vector, transiently expressed by HEK-293E suspension cells, and secreted into the culture media. The final yield after purification and activation was 10-15 mg/L. The enzymatic activity of granzyme B in GrB-Fc-CEA was comparable to that of commercially available human granzyme B. In vitro cytotoxicity against CEA-positive cell lines demonstrated cytotoxicity in the nanomolar range, compared to micromolar range cytotoxicity against control (CEA-negative) cell lines. Importantly, GrB-Fc-CEA cytotoxicity against tumor cells in vitro was unaffected by exogenously-added CEA (200 ng/ml) extracellular domain, suggesting that the construct is not competitively absorbed by soluble CEA released by tumors and present in the circulation of patients. GrB-Fc-CEA internalized rapidly (within one hour) into the target cell lines A-431 and HT-1080/CEA whereas internalization was not observed with untargeted granzyme B, clearly demonstrating receptor-mediated internalization. GrB-Fc-CEA induced mitochondrial depolarization and caspase activation, consistent with the well-described granzyme B mechanism of action. Treatment of female nude mice bearing A-549 tumors with GrB-Fc-CEA (80 mg/kg over 5 doses) resulted in a significant growth inhibition of the established tumors compared to vehicle controls (1200 mm3 vs <40 mm3). Complete tumor regression was observed in seven of ten tumors in mice treated with GrB-Fc-CEA. Mouse weight was unaffected in both treated and control groups for the duration of the study, suggesting no apparent toxicity of the construct. Thus, GrB-Fc-CEA appears to have significant potential in targeting a large category of CEA-expressing solid tumors and is a promising candidate for further pre-clinical development. Research conducted, in part, by the Clayton Foundation for Research. Citation Format: Lawrence H. Cheung, Khalid A. Mohamedali, Ana Alvarez-Cienfuegos, Walter N. Hittelman, Michael G. Rosenblum. An internalizing anti-CEA construct for targeted delivery of granzyme B results in significant antitumor activity in human lung carcinoma xenografts [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 3439.

Abstract 1614: Creation of malignant peripheral nerve sheath tumor models deficient for polycomb repressive complex 2 and identification of therapeutic vulnerabilities

Abstract We constructed models of nerve sheath tumors that arise in Neurofibromatosis Type 1 (NF1) patients and conducted therapeutics discovery using synthetic lethal pharmacogenomic screens. Given both plexiform neurofibromas and malignant peripheral nerve sheath tumors (MPNSTs) arise within the Schwann cell lineage, we developed a drug discovery pipeline to identify targeted therapeutics for treating NF1-related neoplasia. Using CRISPR/Cas9, immortalized human Schwann cell lines were created lacking the NF1 gene or NF1 and components of Polycomb Repressive Complex 2 (PRC2), such as SUZ12. ~80% of all MPNST harbor loss of function mutations in PRC2 genes, which is highly suggestive that perturbation of epigenetic homeostasis plays a role in malignant transformation of neurofibromas. Our models mimic the genetics of MPNSTs and allowed us to identify selectively lethal drugs which exploit vulnerabilities specific to these genetic drivers. We describe the identification of these novel drugs, extensive in vivo testing in models of MPNST, and mechanistic studies to explain why they act specifically on MPNSTs. We identified compounds showing selective lethality towards NF1/SUZ12 double mutant cells. These include drugs affecting epigenetic homeostasis, such as HDAC inhibitors (HDACi). Moreover, many of these drugs showed strong synergy when tested in combination with a MEK inhibitor (MEKi) against NF1/SUZ12 deficient human Schwann and MPNST cell lines. We investigated the mechanism of this synergy using genetics, transcriptome, and proteome analysis. Interestingly, these mechanistic studies revealed possible emerging avenues of resistance the cells are utilizing to compensate for and survive therapeutic intervention. For instance, proteomics analysis of PRC2 deficient MPNST cell lines treated with combination therapy of MEKi and HDACi indicates activation of specific survival and stress response pathways. Targeting these pathways in conjunction with the therapeutics we have identified could prevent emergence of resistance and tumor escape. Clinically interesting drug candidates were advanced and tested as single agents and in combination in multiple in vivo models of MPNST (including patient derived and cell line xenografts). The FDA approved drugs vorinostat (HDACi) and selumetinib (MEKi) have shown dramatic efficacy, with combination therapy exhibiting strong synergy in vivo. This includes durable responses in survival studies and the ability to dramatically shrink established tumors, with treated tumors showing markers of apoptosis. The discovery of novel agents effective against several models of MPNST is exciting as there are currently no approved targeted therapies for MPNSTs. Our results implicate targeting of epigenetic homeostasis, in combination with MEKi, as a major vulnerability of MPNSTs deficient for PRC2 activity. Citation Format: Kyle B. Williams, Alex Larsson, Justin Tibbits, Christopher L. Moertel, David A. Largaespada. Creation of malignant peripheral nerve sheath tumor models deficient for polycomb repressive complex 2 and identification of therapeutic vulnerabilities [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 1614.

Abstract 4214: LIGHT (TNFSF14) costimulation with TIGIT blockade broadens the activity of checkpoint inhibitors (CPIs) into CPI refractory and resistant tumors through targeted myeloid cell and effector lymphocyte activation

Abstract Although increased TIGIT expression on TILs is associated with poor survival in patients with cancer, monotherapy TIGIT antibody blockade has not yet demonstrated significant clinical activity. PD-1 is commonly co-expressed on TILs, and PD-1 mediated SHP-2 signaling inhibits DNAM-1 (CD226), preventing PVR co-stimulation in the setting of TIGIT blockade. The interaction between PD-1 and CD226 explains why TIGIT blockade only translates to clinical benefit in the setting of co-blockade of PD-1/L1, however does not explain why co-blockade of TIGIT and PD-1/L1 does not provide benefit in advanced and PD-1/L1 resistant tumors. In advanced and PD-1/L1 resistant tumors, progressive downregulation of CD226 has been reported. Thus, we hypothesized that the lack of CD226 may underlie the lack of clinical responses to combined TIGIT/PD-1/L1 blockade in PD-1/L1 experienced tumors and we sought to identify alternative costimulatory receptors with high-expression. Analysis of TCGA, as well as single-cell RNASeq from TILs, identified HVEM and LTβR as two costimulatory receptors with higher expression in advanced cancers as compared to CD226. HVEM and LTβR directly activate CD8+ T, natural killer (NK), and myeloid cells when bound by their TNF superfamily ligand, known as LIGHT. LIGHT potentiates effector lymphocyte function through signaling that obviates the co-stimulatory role of DNAM-1. We demonstrated that when the extracellular domain (ECD) of LIGHT is combined with the ECD of TIGIT on a TIGIT-Fc-LIGHT bi-functional fusion protein, the simultaneous blockade of PVR, PVRL2, PVRL3, and Nectin-4 and immune co-stimulation by LIGHT increased CD8+ T and NK infiltration into tumors. This translated into tumor cell killing, regression of established tumors, and improved survival in preclinical models of checkpoint primary and acquired resistance. The translation of TIGIT-Fc-LIGHT activity was evaluated in cynomolgus macaques and was well tolerated at doses up to 40 mg/kg. A series of adaptive immune and proinflammatory cytokines, including IL-2, CCL2, CCL4, IL-10, CXCL10, and CCL17, were induced within two hours of TIGIT-Fc-LIGHT infusion. Additionally, receptor engagement led to the rapid margination of HVEM+CD8+ T cells from the periphery into secondary immune tissues. This network of cytokines and post-dose immune cell margination identified in the monkey, was identical to findings in murine models that ultimately translated into significant anti-tumor responses. Lastly, the combination of TIGIT-Fc-LIGHT with anti-PD(L)1 broadened anti-tumor activity of the checkpoint antibodies in aggressive CPI-resistant tumors. These results suggest that LIGHT could be the differentiator that extends the clinical activity of conventional CPIs into PD-L1 low or CPI acquired resistance tumors. Citation Format: Kyung Jin Yoo, Louis Gonzalez, Kellsey Johannes, Jayalakshmi Miriyala, Karen Lenz, Kristen Campbell, Suresh de Silva, Taylor H. Schreiber, George J. Fromm. LIGHT (TNFSF14) costimulation with TIGIT blockade broadens the activity of checkpoint inhibitors (CPIs) into CPI refractory and resistant tumors through targeted myeloid cell and effector lymphocyte activation [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 4214.

Abstract 1886: Targeting oral squamous cell carcinoma tumor microenvironment through simultaneous modulation of pattern recognition receptors and angiogenic factors using a synthetic polymer-based drug

Abstract Squamous cell carcinomas (SCCs) are the dominant form of cancers of the upper aerodigestive tract. Main risk factors are smoking and human papilloma virus (HPV) infection. Despite the HPV vaccination efforts, the number of oropharyngeal cancers (OSCCs) has been steadily increasing especially in males. Standard of care primarily focuses on the surgical removal of cancerous tissues followed by chemoradiation therapy. However, the recurrence rate of OSCCs remains fairly high as the tumor frequently develops therapy resistance and more effective therapeutic regimens are needed. The chronic inflammatory state yet paradoxical immunosuppressive tumor microenvironment has been regarded as a cause of therapy resistance and suggested as a potential therapeutic target. Pattern recognition receptors (PRRs) such as toll-like receptors (TLRs) contribute to pro-inflammatory tissue responses. TLR2 present in the OSCC tumor appears to play important roles in regulating immune responses and promoting pro-inflammatory tumor microenvironment. GM-1111 is a synthetic glycosaminoglycan-based anti-inflammatory molecule that potently inhibits PRRs such as TLR2 and NLRP3 inflammasome. In addition, GM-1111 inhibits multiple angiogenic molecules - CXCL12, VEGF, and FGF2. We hypothesized that GM-1111 might be able to reduce OSCC tumor growth by simultaneously disrupting pro-inflammatory as well as angiogenic signaling. To test this hypothesis, male CD1 nude mice bearing FaDu cell tumor in the back were treated with either (1) vehicle, (2) GM-1111 (30 mg/kg, q.d., s.c.), (3) vehicle with irradiation (2 Gy/day x 5 days), or (4) GM-1111 with irradiation 17 days. Established tumors grew in two phases from slow for the initial 7 days to fast growths during the remaining days. Irradiation- or drug-induced tumor regression was observable about 5 days after the last radiation therapy (day 10). Compared to the vehicle only (1) treatment group, GM-1111 treated animals (groups 1 vs. 2: p = 0.0032; groups 1 vs. 3: p < 0.0001; groups 1 vs. 4: p < 0.0001) demonstrated significantly reduced tumor growth. These results suggest that GM-1111 has a potential to reduce OSCC and further confirm our previous orthotopic human tongue SCC-25 cell xenograft study data. Citation Format: Won Yong Lee, Frida Ponthan, Stephen T. Sonis. Targeting oral squamous cell carcinoma tumor microenvironment through simultaneous modulation of pattern recognition receptors and angiogenic factors using a synthetic polymer-based drug [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 1886.

Abstract 2749: iPSC-derived natural killer cells as the front-runner program of the EVOcells Oncology platform: From inception to translational validation using patient samples

Abstract Cancer immunotherapy has transformed the oncology landscape and shown promising therapeutic results in the clinic but it is also clearly facing multiple limitations. So far, the focus of immunotherapies has mainly been on the modulation of T-cells. Natural killer (NK) cells offer promising perspectives for immunotherapies as they directly kill tumor cells and produce pro-inflammatory cytokines to activate and recruit T-cells and other immune cells. Thus, NK cells are considered key players in the anti-tumor immune response that can make a “cold” tumor “hot”. However, in the tumor microenvironment (TME) of cancer patients, the low numbers of NK cells present and their exhaustion due to local immunosuppression often limit tumor cell killing. The development of NK based cell therapy products can overcome these limitations, either as single therapy or in combination with biologics such as e.g. NK cell engagers or monoclonal antibodies for boosting ADCC (antibody dependent cell cytotoxicity). To overcome the main hurdle of current autologous cell therapies, the EVOcells Oncology platform aims at developing a streamlined manufacturing process for iPSC-derived immune effector cells. The goal is to develop “off-the-shelf” cell therapy products for clinical use in large patient populations. We are currently establishing a multi-asset portfolio combining multiple immune effector cell types with different genetic editing strategies that serve to optimize this approach further. iPSC-derived NK cells (iNK cells) offer an attractive option for improving both therapeutic efficacy and safety of CAR cell therapies. Our robust, feeder-free production protocol produces iNK cells that can be reliably frozen with good recovery rates. We have generated multiple genetically modified iNK cells and have validated that the genetic modifications boost iNK function. At the end of the production process, iNK cells show phenotypic properties and single cell RNA sequencing profiles comparable to blood-derived NK cells. Importantly, iNK cells are fully functional with the ability to produce key pro-inflammatory cytokines and to form lytic immunological synapses leading to efficient killing of cancer cell lines. In addition to the killing of established tumor cell lines, we also evaluated the ability of iNK cells to kill primary leukemia cells ex vivo by using blood samples from B-cell chronic lymphocytic leukemia patients. In combination with a standard of care anti-CD20 antibody, iNK cells demonstrated the ability to kill primary leukemia cells via ADCC. Taken together, these promising results obtained with iNK cells show the potential of the EVOcells Oncology platform to deliver the next generation of cell therapies in oncology paving the way to develop a portfolio of iPSC-derived immune effector cells. Citation Format: Michael Esquerré, Audrey Holtzinger, Mélanie Pichery, Stefanie Pfaender, Saniye Yumlu, Mandy Richter, Delphine Betous, Oriane Bombarde, Mylène Gador, Nadja Sailer, Michael Paillasse, Loïc Ysebaert, Fabien Despas, Matthias Austen, Andreas Scheel, Markus Dangl. iPSC-derived natural killer cells as the front-runner program of the EVOcells Oncology platform: From inception to translational validation using patient samples [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 2749.

Abstract 4246: ABL103, A novel T-cell engaging bispecific antibody, exhibits potent in vitro and vivo antitumor activity and low toxicity via B7-H4 dependent 4-1BB activation in tumor microenvironment

Abstract B7-H4 (B7x, VTCN1), a member of the B7-family, is overexpressed in majority of cancer patients with ovarian, endometrial and breast cancers. B7-H4 expression was also observed in tumor associated macrophages and implicated as an immune checkpoint regulating T cell responses. Although B7-H4 expression in normal tissues is quite limited, tumor-specific immune response by ABL103 would minimize potential adverse effects. ABL103, B7-H4 Grabody-T, is a First-in Class bispecific antibody targeting B7-H4 and 4-1BB and augments T cell function by a dual mechanism, i.e., 1) blocks the B7-H4 mediated T cell inhibition, and 2) elicits superior T cell activation through B7-H4-dependent 4-1BB clustering. Our data showed that simultaneous binding of B7-H4 and 4-1BB by ABL103 led to potent in vitro T cell activation only in the presence of B7-H4 expressing tumor cells. In the established tumor model, ABL103 potently inhibited tumor progression in a dose-dependent manner and showed higher rate of complete remission (CR) at 2 and 10 mg/kg dose groups. Moreover, mice were protected from the tumor re-challenge 3 months after cessation of ABL103 treatment, suggesting long-term memory has been established. In 4-week pilot tox study using cynomolgus monkeys, ABL103 was tolerable up to 100 mg/kg dosed weekly with no ABL103-related toxicity observations. To understand the relationship of target expression, expression of B7-H4, CD4, CD8, PD-L1, and 4-1BB was examined in 142 ovarian cancer patient biopsies. About 83% of ovarian cancer patient tissues showed B7-H4 positive staining and 62% of them showed strong positive. In addition, the staining of 4-1BB as well as CD4 and CD8 T cells were observed in both tumor nest and stroma, suggesting that cross-linking of B7-H4 and 4-1BB is feasible in tumor microenvironment. Overall, ABL103 has a strong in vitro and vivo anti-tumor activity and good safety profile via B7-H4-dependent 4-1BB activation. This strongly suggests ABL103 is a promising therapeutic agent potentially benefitting patients with B7-H4 overexpression. Citation Format: Kyungjin Park, Yangsoon Lee, Saeyi Lim, Kyeongsu Park, Eunjung Kim, Hanbyul Lee, Jiseon Yoo, Youngdon Pak, Yeunju Kim, Minji Ko, Jonghwa Won. ABL103, A novel T-cell engaging bispecific antibody, exhibits potent in vitro and vivo antitumor activity and low toxicity via B7-H4 dependent 4-1BB activation in tumor microenvironment [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 4246.

CASTING: A Potent Supramolecular Strategy to Cytosolically Deliver STING Agonist for Cancer Immunotherapy and SARS-CoV-2 Vaccination

CASTING: A Potent Supramolecular Strategy to Cytosolically Deliver STING Agonist for Cancer Immunotherapy and SARS-CoV-2 Vaccination

Intratumoural administration of an NKT cell agonist with CpG promotes NKT cell infiltration associated with an enhanced antitumour response and abscopal effect

ABSTRACT Intratumoural administration of unmethylated cytosine-phosphate-guanine motifs (CpG) to stimulate toll-like receptor (TLR)-9 has been shown to induce tumour regression in preclinical studies and some efficacy in the clinic. Because activated natural killer T (NKT) cells can cooperate with pattern-recognition via TLRs to improve adaptive immune responses, we assessed the impact of combining a repeated dosing regimen of intratumoural CpG with a single intratumoural dose of the NKT cell agonist α-galactosylceramide (α-GalCer). The combination was superior to CpG alone at inducing regression of established tumours in several murine tumour models, primarily mediated by CD8+ T cells. An antitumour effect on distant untreated tumours (abscopal effect) was reliant on sustained activity of NKT cells and was associated with infiltration of KLRG1+ NKT cells in tumours and draining lymph nodes at both injected and untreated distant sites. Cytometric analysis pointed to increased exposure to type I interferon (IFN) affecting many immune cell types in the tumour and lymphoid organs. Accordingly, antitumour activity was lost in animals in which dendritic cells (DCs) were incapable of signaling through the type I IFN receptor. Studies in conditional ablation models showed that conventional type 1 DCs and plasmacytoid DCs were required for the response. In tumour models where the combined treatment was less effective, the addition of tumour-antigen derived peptide, preferably conjugated to α-GalCer, significantly enhanced the antitumour response. The combination of TLR ligation, NKT cell agonism, and peptide delivery could therefore be adapted to induce responses to both known and unknown antigens.

YAP1 is essential for malignant mesothelioma tumor maintenance

Malignant pleural mesothelioma, a tumor arising from the membrane covering the lungs and the inner side of the ribs, is a cancer in which genetic alterations of genes encoding proteins that act on or are part of the Hippo-YAP1 signaling pathway are frequent. Dysfunctional Hippo signaling may result in aberrant activation of the transcriptional coactivator protein YAP1, which binds to and activates transcription factors of the TEAD family. Recent studies have associated elevated YAP1 protein activity with a poor prognosis of malignant mesothelioma and its resistance to current therapies, but its role in tumor maintenance is unclear. In this study, we investigate the dependence of malignant mesothelioma on YAP1 signaling to maintain fully established tumors in vivo. We show that downregulation of YAP1 in a dysfunctional Hippo genetic background results in the inhibition of YAP1/TEAD-dependent gene expression, the induction of apoptosis, and the inhibition of tumor cell growth in vitro. The conditional downregulation of YAP1 in established tumor xenografts leads to the inhibition of YAP1-dependent gene transcription and eventually tumor regression. This effect is only seen in the YAP1-activated MSTO-211H mesothelioma xenograft model, but not in the Hippo-independent HCT116 colon cancer xenograft model. Our data demonstrate that, in the context of a Hippo pathway mutated background, YAP1 activity alone is enough to maintain the growth of established tumors in vivo, thus validating the concept of inhibiting the activated YAP1-TEAD complex for the treatment of malignant pleural mesothelioma patients.

Non-cytotoxic nanoparticles re-educating macrophages achieving both innate and adaptive immune responses for tumor therapy

Macrophages are important antigen-presenting cells to combat tumor via both innate and adaptive immunity, while they are programmed to M2 phenotype in established tumors and instead promote cancer development and metastasis. Here, we develop a nanomedicine that can re-educate M2 polarized macrophages to restore their anti-tumor activities. The nanomedicine has a core-shell structure to co-load IPI549, a PI3Kγ inhibitor, and CpG, a Toll-like receptor 9 agonist. Specifically, the hydrophobic IPI549 is self-assembled into a pure drug nano-core, while MOF shell layer is coated for CpG encapsulation, achieving extra-high total drugs loading of 44%. Such nanosystem could facilitate intracellular delivery of the payloads but without any cytotoxicity, displaying excellent biocompatibility. After entering macrophages, the released IPI549 and CpG exert a synergistic effect to switch macrophages from M2 to M1 phenotype, which enables anti-tumor activities via directly engulfing tumor cells or excreting tumor killing cytokines. Moreover, tumor antigens released from the dying tumor cells could be effectively presented by the re-educated macrophages owing to the up-regulation of various antigen presenting mediators, resulting in infiltration and activation of cytotoxic T lymphocytes. As a result, the nanosystem triggers a robust anti-tumor immune response in combination with PD-L1 antibody to inhibit tumor growth and metastasis. This work provides a non-cytotoxic nanomedicine to modulate tumor immune microenvironment by reprograming macrophages.

Diagnostic Potential of Exosomal microRNAs in Colorectal Cancer.

Background: Despite the significance of colonoscopy for early diagnosis of colorectal adenocarcinoma (CRC), population-wide screening remains challenging, mainly because of low acceptance rates. Herein, exosomal (exo-miR) and free circulating microRNA (c-miR) may be used as liquid biopsies in CRC to identify individuals at risk. Direct comparison of both compartments has shown inconclusive results, which is why we directly compared a panel of 10 microRNAs in this entity. Methods: Exo-miR and c-miR levels were measured using real-time quantitative PCR after isolation from serum specimens in a cohort of 69 patients. Furthermore, results were compared to established tumor markers CEA and CA 19-9. Results: Direct comparison of exo- and c-miR biopsy results showed significantly higher microRNA levels in the exosomal compartment (p < 0.001). Exo-Let7, exo-miR-16 and exo-miR-23 significantly differed between CRC and healthy controls (all p < 0.05), while no c-miR showed this potential. Sensitivity and specificity can be further enhanced using combinations of multiple exosomal miRNAs. Conclusions: Exosomal microRNA should be considered as a promising biomarker in CRC for future studies. Nonetheless, results may show interference with common comorbidities, which must be taken into account in future studies.

Synthetic libraries of immune cells displaying a diverse repertoire of chimaeric antigen receptors as a potent cancer immunotherapy.

Cancer immunotherapies rely on one or few specific tumour-associated antigens. However, the adaptive immune system relies on a large and diverse repertoire of antibodies for antigen recognition. Here we report the development and applicability of libraries of immune cells displaying diverse repertoires of chimaeric antigen receptors (CARs) that can recognize non-self antigens and display antigen-dependent clonal expansion, with the expanded population of tumour-specific effector cells leading to long-lasting antitumour responses in mouse models of epithelial tumours. The intravenous injection of synthetic libraries of murine CARs on TET2- T cells led to robust immunological memory and the recognition of mutated or evolved tumours, owing to the maintenance of CAR diversity. Off-the-shelf libraries of 106 murine or human CAR clones displayed on genetically modified human NK-92 cancer cells completely eliminated established tumours in mice with murine xenografts and patient-derived xenografts. Synthetically generated CAR libraries may aid the discovery of new CARs and the development of immunotherapies.

IMMU-23. Novel gene-edited CAR-T cell therapy against Diffuse Intrinsic Pontine Glioma

BACKGROUND: We identified high expression of CD99 in DIPG tumors and developed a CAR using our newly identified single chain variable fragment (scFv) targeting CD99 incorporating a 4-1BB co-stimulatory domain. This CD99 CAR demonstrated the ability to dramatically shrink the established orthotopic DIPG tumor, however tumor recurrence remains an obstacle to cure, due to a loss of the CAR-T cells as they also express the target antigen, CD99 (fratricide). To overcome this obstacle, we modified these CAR-T by editing out CD99. METHODS: CD99 was knocked-out from the human T cells using CRISPR-cas9 gene-editing and subsequently transduced with our CD99 CAR-encoding virus, and isolated the pure population of CD99KO T-cells. These novel, gene-edited T-cells expressing CD99 CAR (“CD99KO CARs”) and the un-edited ones (“CD99 CAR”) were tested for tumor-lysis function when co-cultured with DIPG cells. DIPG tumor-bearing mice infused with a one-time dose of CD99KO CAR-T cells or CD99 CAR- or CD19 control CAR-T cells and were monitored for changes in the tumor burden. At the endpoint spleen and bone marrow were isolated to test for CAR+ cell persistence. RESULTS: The CD99KO CAR-T cells demonstrated effective tumor-lysis when co-cultured with DIPG cells. CD99KO CAR-T cells targeting CD99 showed complete clearance of DIPG tumor in orthotopic DIPG mouse models, and no tumor recurrence was seen well-beyond the time frame of expected tumor recurrence after treatment with un-edited CD99 CAR-T cells. There was an un-precedented increase in the xenograft survival, > 200 days, in mice treated with CD99KO CARs and at which time point sustained persistence of CAR+ cells were evident in the animal spleen and bone marrow. CONCLUSIONS: We have generated a new and promising CAR-T cell therapy that is effective against DIPG with enhanced persistence in animal models which is critical for clinical translation.

MODL-12.Ex vivo model identifies druggable vulnerabilities in medulloblastoma

Abstract Medulloblastoma (MB) is the most common malignant brain tumour in children and long-term sequelae in survivors caused by the harsh, non-targeted treatments is common. Current screening protocols for the detection of novel druggable vulnerabilities rely on 2D and 3D cell culture systems using established or primary tumour cells. However, these assays do neither provide the cellular, chemical and biophysical context of the tissue, nor reveal drug effects on the invasive behavior of the tumour cells. To overcome these limitations, we have established ex vivo Organotypic Cerebellum Slice Co-culture (OCSC) models for SHH and group 3 MB tumours, which provide spatio-temporal insights in the growth and dissemination behaviour of the tumour cells. OCSCs represent an excellent translational model for patient sample profiling, drug testing, and microenvironmental or explorative target identification studies in a physiologically relevant tissue context. We found that the AURKB inhibitor Barasertib (AZD-1152), which was ineffective to reduce ONS-76 cell invasion in vitro, effectively reduced tumour volume and proliferation of these SHH tumour cells in the tissue context. The AURKB inhibitor thus displays higher efficacy ex vivo, indicating an impact of the microenvironment on its functional activity. We further validated tumour suppressive activity of AURKB inhibition with Barasertib at low nanomolar concentration or with siRNA-mediated depletion of AURKB in Grp3 MB tumour cells and in primary tumour cells ex vivo, thereby confirming the therapeutic potential of AURKB inhibition against MB progression. We furthermore found that the combination of AURKB inhibition with irradiation or with the SRC inhibitor Dasatinib, lead to a near complete eradication of the tumour cells. Thus, OCSCs are a clinically relevant model where tumour material can be efficiently tested for drug sensitivities, which allowed us to identify AURKB inhibition as an effective treatment against tissue invasive MB.

HGG-59. Pediatric high-grade gliomas and the WHO classification on CNS Tumors - Different perspectives of pediatric neuro-oncologists and neuropathologists in the light of recent updates

Abstract BACKGROUND: The WHO Classification of Tumors of the Central Nervous System has undergone major restructuring following rapid advances in brain tumor genomics and epigenomics. The most significant changes resulted from the introduction of molecularly defined diagnostic criteria in 2016 (revised 4th edition). In 2021 (5th edition), further essential molecular criteria were incorporated. In the present study, we sought to investigate potential differences between specialists in perception of these newly defined molecular subtypes of pediatric high-grade gliomas (pedHGG). METHODS: We designed a 22-question survey studying the impact of the revised 4th edition of the WHO classification on pedHGG. Data were collected and statistically analyzed to capture the spectrum of viewpoints and possible differences among neuro-oncologists and neuropathologists. RESULTS: 465 participants from 53 countries responded, of which 187 pediatric neuro-oncologists (40%), 160 neuropathologists (34%) and 118 experts in other related fields (neurosurgeons, radiotherapists, neuroradiologists and others; 26%). Neuro-oncologists reported having issues with the introduction of new molecular entities, such as the abolishment and renaming of established tumor entities. Neuropathologists did not define these problems to the same extent. However, both groups felt that in the 2016 version, less relevant or insufficient diagnostic definitions were available for pedHGG. Within the 2021 WHO classification, a substantial improvement was perceived regarding the definition of pedHGG entities. However, some issues of high clinical relevance, like the definition of clinical phenotypes such as diffuse intrinsic pontine glioma (DIPG) and gliomatosis cerebri, are yet to be addressed. CONCLUSIONS: Within the WHO classification of pediatric brain tumors, such as high-grade gliomas, rapid changes in nomenclature have been introduced because of substantial improvement in molecular characterization. This study highlights that ongoing cross-talk between advancing classification of pedHGG subtypes and its biological relevance and clinical impact is essential.

Trastuzumab Deruxtecan, Antibody-Drug Conjugate Targeting HER2, Is Effective in Pediatric Malignancies: A Report by the Pediatric Preclinical Testing Consortium.

HER2 is expressed in many pediatric solid tumors and is a target for innovative immune therapies including CAR-T cells and antibody-drug conjugates (ADC). We evaluated the preclinical efficacy of trastuzumab deruxtecan (T-DXd, DS-8201a), a humanized monoclonal HER2-targeting antibody conjugated to a topoisomerase 1 inhibitor, DXd, in patient- and cell line-derived xenograft (PDX/CDX) models. HER2 mRNA expression was determined using RNA-seq and protein expression via IHC across multiple pediatric tumor PDX models. Osteosarcoma (OS), malignant rhabdoid tumor (MRT), and Wilms tumor (WT) models with varying HER2 expression were tested using 10 mice per group. Additional histologies such as Ewing sarcoma (EWS), rhabdomyosarcoma (RMS), neuroblastoma (NB), and brain tumors were evaluated using single mouse testing (SMT) experiments. T-DXd or vehicle control was administered intravenously to mice harboring established flank tumors at a dose of 5 mg/kg on day 1. Event-free survival (EFS) and objective response were compared between treatment and control groups. HER2 mRNA expression was observed across histologies, with the highest expression in WT (median = 22 FPKM), followed by MRT, OS, and EWS. The relationship between HER2 protein and mRNA expression was inconsistent. T-DXd significantly prolonged EFS in 6/7 OS, 2/2 MRT, and 3/3 WT PDX models. Complete response (CR) or maintained CR (MCR) were observed for 4/5 WT and MRT models, whereas stable disease was the best response among OS models. SMT experiments also demonstrated activity across multiple solid tumors. Clinical trials assessing the efficacy of a HER2-directed ADC in pediatric patients with HER2-expressing tumors should be considered.

Regression of EGFR positive established solid tumors in mice with the conditionally active T cell engager TAK-186

BackgroundDespite clinical success with T cell engagers (TCEs) targeting hematological malignancies, achieving a safe and efficacious dose in patients with solid tumors remains challenging. Due to potency, low levels of...

MiR-198 sensitizes pancreatic cancer to gemcitabine treatment through downregulation of VCP-mediated autophagy maturation.

e16290 Background: The mechanism of human pancreatic ductal adenocarcinoma (PDAC) resistance to nucleoside analog and first-line chemotherapy drug gemcitabine is not clearly understood, but some studies have associated it with increased autophagy. In cancer biology, autophagy plays dual roles as it can promote tumor suppression during early stages, but in established tumors, it plays a crucial role in tumor growth by enhancing survival under metabolic and therapeutic stress. We have previously found that miR-198 acts as a tumor suppressor in PDAC through the targeting of a network of tumorigenic factors, including the Valosin-containing protein (VCP), which has been reported to play an important role in autophagy. In this study, we investigate whether the repression of VCP through miR-198 replacement disrupts the autophagy process and sensitizes PDAC cells to gemcitabine treatment. Methods: MIAPaCa2 cells with forced overexpression of mesothelin (MSLN) and AsPC-1 cell lines and, CDX and PDX mouse models were used for gemcitabine sensitization studies. For miR-198 replacement, a miR-198 expression vector-loaded lactic co-glycolic-acid-modified polyethylenimine polyplex (LPNP-p198) was used. Autophagy disruption experiments were run over miR-198 and/or VCP overexpressing cell lines. Nude mice were used for subcutaneous and orthotopic CDX models and SCID/Beige were used for the subcutaneous PDX model. Results: Cell lines were treated with LPNP-p198 in combination with gemcitabine and cell growth was significantly inhibited when compared to gemcitabine alone. Additionally, it was determined that miR-198 disrupts the autophagy maturation process and, that it is then restored by overexpression of VCP. LPNP-p198 can effectively enter tumor cells and induce tumor sensitization in vivo, resulting in an 80-90% reduction of tumor burden and metastatic spread in the LPNP-p198 plus gemcitabine group when compared to controls, with the concomitant downregulation of VCP expression in the tumor tissue. In addition, this group had the fewest mice with jaundice, ascites, and metastases to the abdominal cavity, spleen, liver, and kidney. Finally, to address the potential of the observed effect in the context of the heterogenic nature of PDAC, we confirmed our findings in a PDX mouse model, where a marked reduction in tumor burden was observed in the LPNP-p198 plus gemcitabine group compared to controls. Conclusions: Our findings indicate that miR-198 replacement disrupts the autophagy maturation process and sensitizes PDAC cells to gemcitabine through VCP repression, indicating a potential therapeutic strategy for targeting gemcitabine-resistant PDAC and, establishing the use of LPNPs as a prototype for effective nucleic acid delivery in vitro and in vivo, with potential to be used from bench to clinic.

Targeting EP2 receptor with multifaceted mechanisms for high-risk neuroblastoma.

SUMMARYProstaglandin E2 (PGE2) promotes tumor cell proliferation, migration, and invasion, fostering an inflammation-enriched microenvironment that facilitates angiogenesis and immune evasion. However, the PGE2 receptor subtype (EP1–EP4) involved in neuroblastoma (NB) growth remains elusive. Herein, we show that the EP2 receptor highly correlates with NB aggressiveness and acts as a predominant Gαs-coupled receptor mediating PGE2-initiated cyclic AMP (cAMP) signaling in NB cells with high-risk factors, including 11q deletion and MYCN amplification. Knockout of EP2 in NB cells blocks the development of xenografts, and its conditional knockdown prevents established tumors from progressing. Pharmacological inhibition of EP2 by our recently developed antagonist TG6-129 suppresses the growth of NB xenografts in nude mice and syngeneic allografts in immunocompetent hosts, accompanied by anti-inflammatory, antiangiogenic, and apoptotic effects. This proof-of-concept study suggests that the PGE2/EP2 signaling pathway contributes to NB malignancy and that EP2 inhibition by our drug-like compounds provides a promising strategy to treat this deadly pediatric cancer.

Optimal target saturation of ligand-blocking anti-GITR antibody IBI37G5 dictates FcγR-independent GITR agonism and antitumor activity

SummaryGlucocorticoid-induced tumor necrosis factor receptor (GITR) is a co-stimulatory receptor and an important target for cancer immunotherapy. We herein present a potent FcγR-independent GITR agonist IBI37G5 that can effectively activate effector T cells and synergize with anti-programmed death 1 (PD1) antibody to eradicate established tumors. IBI37G5 depends on both antibody bivalency and GITR homo-dimerization for efficient receptor cross-linking. Functional analyses reveal bell-shaped dose responses due to the unique 2:2 antibody-receptor stoichiometry required for GITR activation. Antibody self-competition is observed after concentration exceeded that of 100% receptor occupancy (RO), which leads to antibody monovalent binding and loss of activity. Retrospective pharmacokinetics/pharmacodynamics analysis demonstrates that the maximal efficacy is achieved at medium doses with drug exposure near saturating GITR occupancy during the dosing cycle. Finally, we propose an alternative dose-finding strategy that does not rely on the traditional maximal tolerated dose (MTD)-based paradigm but instead on utilizing the RO-function relations as biomarker to guide the clinical translation of GITR and similar co-stimulatory agonists.