Dose Range Research Articles

Abstract 2475: MB5029: A novel anti-PD-1 x IL-2v with improved safety profile and enhanced anti-tumor efficacy by selectively targeting activated T cells

Abstract Interleukin-2 (IL-2) offers therapeutic potential for cancer treatment, however, its clinical application is limited due to systemic adverse effects, such as vascular leakage syndrome (VLS). Notably, the high affinity of IL-2 for the IL-2 receptor α (IL-2Rα) activates immune-suppressive Treg cells and endothelial cells, leading to undesired outcomes. To address this challenge, a number of clinical studies are under development including non-α IL-2 variant (IL-2v) drugs and further the anti-PD-1 x IL-2v for targeted delivery of its IL-2v to PD-1 enriched T cells. Nevertheless, concerns about peripheral toxicity have not yet been solved. Thus, here we present MB5029, a novel anti-PD-1 x IL-2v bispecific fusion protein with modulated IL-2R binding affinity to mitigate the peripheral toxicity maintaining the anti-tumor efficacy. MB5029 has unique characteristics with no binding affinity to IL-2Rα and reduced binding affinity to IL-2Rγ, which is widely and highly expressed in various immune cells. MB5029 exhibited strong STAT5 phosphorylation activity under activated conditions but weak activity in the resting conditions, while the anti-PD-1 x IL-2v (non-α) showed strong STAT5 phosphorylation in both resting and activated conditions. In addition, MB5029 strongly induced the expansion of CD8+ T cells in the activated conditions but less expansion of CD8+ T cells in the resting conditions, in contrast to anti-PD-1 x IL-2v (non-α). The murine surrogate of MB5029 showed remarkable anti-tumor efficacy without body weight loss at the dose range of 1 to 10 mg/kg, whereas anti-muPD-1 x IL-2v (non-α) showed significant body weight loss in several tumor-bearing mice models. Moreover, MB5029 showed 100% complete regression (6/6) at the dose of 5 mg/kg or higher in the MC38 tumor model. The mice with complete response showed no signs of relapse upon tumor re-challenge until day 56, indicating a durable memory response. MB5029 exhibited a comparatively superior safety profile in normal mice. While anti-muPD-1 x IL-2v (non-α) led to significant body weight loss at the dose of 5 mg/kg in normal mice, the murine surrogate of MB5029 was shown to be well-tolerated up to a dose of 30 mg/kg without body weight loss. MB5029 exhibited dose-proportional PK properties and induced expansion of CD8+ T cells in a dose-dependent manner in cynomolgus monkeys. MB5029 is shown to be well-tolerated up to 9 mg/kg without severe clinical signs and no significant increase in eosinophil and cytokines in the blood. Overall, MB5029 represents a promising potential as an immunotherapeutic approach, as it has demonstrated both strong efficacy and good tolerability, along with improved safety margins. Citation Format: Bom Park, Hyojoo Bang, So Woon Kim, Suna Kim, Sunjung Cho, Suyoon Kim, Minwoo Kim, Youngjin Park, Jun-Eui Park. MB5029: A novel anti-PD-1 x IL-2v with improved safety profile and enhanced anti-tumor efficacy by selectively targeting activated T cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2475.

Abstract 394: Preclinical evaluation of ERX-208, a potent inducer of ER stress for the treatment of ovarian cancer

Abstract Background: Ovarian cancer (OCa) is the deadliest kind of gynecologic cancer in the United States. The long-term survival rate for OCa is less than 20% after five years. Intra-tumoral and inter-tumoral heterogeneity is implicated in tumor resistance to conventional therapies and the unsatisfactory clinical outcomes. Addressing these issues necessitates innovative therapies that target intrinsic common vulnerabilities within OCa. Recent studies have highlighted that high basal level of endoplasmic reticulum stress (ERS) in OCa as a critical vulnerability. We have identified a promising compound, ERX-208 that potently induces ERS in cancer cells. The objective of this study is to characterize the mechanisms and activity of ERX-208 using preclinical models. Methods: The biological activity of ERX-208 was examined across 17 distinct OCa cells, representing 5 diverse OCa subtypes. Mechanistic studies utilized Western blotting, immunohistochemistry (IHC), RNA-Seq analysis, and CRISPR/Cas9 knockouts (KO). Pharmacokinetics (PK) and toxicity studies were performed on C57BL/6 mice. Comprehensive preclinical assessments were carried out through cell line-derived xenografts (CDXs), patient-derived xenografts (PDXs), organoids (PDOs) and explants (PDEs). Results: ERX-208 demonstrated an IC50 of approximately 50-100 nM inducing ERS and reducing cell viability in OCa cells. Conversely, normal ovarian surface epithelial cells exhibited minimal effects from ERX-208. In vitro experiments revealed strong ERX-208-induced apoptosis in OCa cell lines. Mechanistic studies employing RNA sequencing, Western blotting, and RT-qPCR, confirmed activation of ERS pathways observed as early as 5 hours post-ERX-208 treatment. Our studies identified LIPA as a potential target, and KO of LIPA significantly diminished ERX-208 activity. Moreover, LIPA KO substantially impeded in vivo OCa tumor growth. ERX-208 up to a dose of 25 mg/kg showed no observable organ toxicity and had no effect on the mice's body weight. Dose range studies identified 10 mg/kg intraperitoneal as the minimal effective dose, achieving more than 50% tumor reduction. In preclinical models, ERX-208 inhibited the growth of OCa CDXs, PDXs, and ex vivo PDEs and PDO’s. IHC analyses indicated reduced proliferation (Ki-67) and increased activation of ERS markers, such as GRP78 and p-PERK. Conclusions: Collectively, our findings underscore the preclinical promise of ERX-208 as a potential therapeutic agent for treating OCa. Conflict: The patents surrounding ERX-208 are licensed to EtiraRx. Citation Format: Suryavathi Viswanadhapalli, Tae-Kyung Lee, Scott Elmore, Gaurav Sharma, Rahul Gopalam, Karla Parra, Tanner Reese, Michael Hsieh, Uday P. Pratap, Xue Yang, Behnam Ebrahimi, Henry Neal, Chia-Yuan Chen, Kara Kassees, Christian Cervantes, Zhenming Xu, Edward Kost, Gangadhara Reddy Sareddy, Rajeshwar R. Tekmal, Jung-Mo Ahn, Ganesh V. Raj, Ratna K. Vadlamudi. Preclinical evaluation of ERX-208, a potent inducer of ER stress for the treatment of ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 394.

Abstract 5302: CB699: A novel mesothelin-binding Humabody® CD40 and CD137 dual-agonist for enhancing immune cell responses against MSLN+ tumors

Abstract INTRODUCTION: CD137 is a TNF receptor superfamily (TNFRSF9) costimulatory receptor expressed by T cells and NK cells. Clustering-mediated CD137 signaling enhances immune cell survival, proliferation, cytokine production and memory formation. CD40 (previously TNFRSF5) is a related superfamily member expressed primarily on B cells and antigen-presenting cells (APCs) which upon clustering is also a key modulator of T cell responses. Mesothelin (MSLN) is highly upregulated on tumor cells in ovarian and pancreatic cancer and select other solid tumour histologies. To avoid systemic toxicity, CB699 is designed to be immunologically active in the presence of any two of its three binding targets, thus enhancing both immune-immune and tumour-immune crosstalk. We present here the preclinical pharmacology of CB699. EXPERIMENTAL PROCEDURES: Binding of CB699 to CD40, CD137 or MSLN was measured by surface plasmon resonance and by flow cytometry. CD137 and/or CD40 agonism, immune cell activation and cytokine secretion were evaluated in vitro using reporter gene assays and primary cell coculture assays in the presence/absence of MSLN-expressing tumor cells. The ability of CB699 to enhance tumor cell killing was characterized in 3D spheroid cocultures. CB699 toxicology and toxicokinetics was assessed in a dose range-finding study in cynomolgus macaques. CB699 was also assessed in vivo with a human CD34+ cell engrafted mouse model. RESULTS: CB699 binds to primary and immortalized cells that express CD40, CD137 or MSLN, with low nanomolar affinity. CB699 also binds to cells from primary disaggregated human solid tumors. CB699 induces CD137 reporter cell activity in cocultures containing cells expressing either CD40 or MSLN. Additionally, CB699 induces CD40 reporter cell activity in cocultures containing cells expressing either CD137 or MSLN. CB699 induces IL-2 secretion and upregulation of CD86 on B cells in primary cocultures containing healthy human PBMCs and MSLN expressing cancer cells. Enhanced tumor cell killing was observed in spheroid cultures containing CB699 and was coupled with immune cell expansion. CB699 showed significant pharmacology in vivo with immune cell engrafted mice. Finally, CB699 was well tolerated in a dose range finding non-human primate toxicology study. CONCLUSIONS: CB699 is a novel MSLN-binding CD137 and CD40 dual agonist that selectively enhances immune cell activity by enhancing both T cell and APC signaling axes. CB699 is positioned to enter clinical development. Citation Format: Andrew J. Pierce, Phillip M. Brailey, Sophie Archer, Holly Spencer, Minjung Song, Timothy Wong, Phillip D. Bartlett, Kieran Williams, Lukas Jasaitis, Philip Bland-Ward. CB699: A novel mesothelin-binding Humabody® CD40 and CD137 dual-agonist for enhancing immune cell responses against MSLN+ tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5302.

Abstract 5299: A novel anti-4-1BB antibody with no liver toxicity and its application in a bi-specific antibody

Abstract 4-1BB (CD137, TNFRSF9) is a co-stimulatory receptor initially identified on T cells, belonging to the tumor necrosis factor receptor superfamily. Primarily expressed on activated CD8+ T cells, it plays a crucial role in immune activation. Moreover, the intracellular domain of 4-1BB is leveraged to enhance the proliferation and cytotoxicity of Chimeric Antigen Receptor-T (CAR-T) cells. However, the development of 4-1BB agonist antibodies has encountered challenges, with limited clinical efficacy (e.g., utomilumab) or dose-dependent liver toxicity (e.g., urelumab). Consequently, there is a significant demand for the development of next-generation 4-1BB agonist antibodies, leading to numerous ongoing preclinical R&D efforts and clinical trials. Here, we introduce a novel fully humanized anti-4-1BB antibody, HLX25, and discuss the development of HLX34, a bispecific antibody targeting both 4-1BB and Her2. HLX25 boasts a unique binding epitope distinct from first-generation 4-1BB antibodies and demonstrates multiple cross-species reactivities. Additionally, HLX25 incorporates an engineered Fc region to enhance intratumoral clustering while reducing toxicity. In vitro studies reveal that HLX25 exhibits activity similar to natural 4-1BB ligands. In contrast, utolimumab shows inadequate activity, and urelumab displays superior activity, suggesting a correlation between efficacy and safety. As reported previously, (As been profiled in our Lab,) HLX25 exhibits robust dose-dependent anti-tumor activity in the MC38/h4-1BB KI mouse model. Noteworthy is the superior safety profile of HLX25 in the h4-1BB KI model, where both HLX25 and utolimumab induce no significant AST and ALT upregulation, unlike urelumab, which demonstrates such upregulation in vivo. A comparative analysis of HLX25 with other second-generation anti-4-1BB antibodies in clinical trials in vivo reveals that HLX25 provides superior tumor inhibition without observed toxicity within the effective dose range. Furthermore, we've developed HLX34, a Her2x4-1BB bispecific antibody, derived from Trastuzumab and HLX25. Its format underwent optimization using a reporter assay, indicating superior in vitro activity. Validation through a cytokine release assay with primary PBMCs in vitro and confirmed in vivo activity in animal models, without observed toxicity, strongly supports the efficacy and safety of HLX34 in Her2-positive tumors. These results provide evidence that our novel anti-4-1BB antibody activates proper 4-1BB signaling through tumor-enriched FcgRIIB or tumor-associated antigen-mediated clustering, inhibiting tumor growth with good safety in both in vitro and in vivo settings. This underscores HLX34 as a promising alternative therapeutic strategy for next-generation cancer immunotherapy. Citation Format: Jen-Kuan Chang, Jie Xue, Chen Dong, An Ju, Toya Baral, Julie Yoo, Yanling Wang, Marco Muda, Qiang Liu, Wenfeng Xu, Weidong Jiang, Lixin Feng. A novel anti-4-1BB antibody with no liver toxicity and its application in a bi-specific antibody [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5299.

Abstract 49: LYL119, an investigational ROR1-targeted CAR T-cell product incorporating four novel reprogramming technologies designed for effective cell therapy for solid tumors

Abstract Effective solid tumor cell therapy requires new strategies to improve T-cell activation, persistence, and durable function. We developed four complementary T-cell reprogramming technologies to enhance chimeric antigen receptor (CAR) T-cell therapy in solid tumors: 1) overexpression of the activator protein 1 (AP-1) family transcription factor c-Jun to delay T-cell exhaustion and improve antitumor activity; 2) nuclear receptor subfamily 4A member 3 (NR4A3) gene knockout (KO) to further delay exhaustion and enhance functionality; 3) Epi-RTM manufacturing protocols to preserve stem-like characteristics; and 4) Stim-RTM technology, a novel activating reagent to improve product potency compared with standard reagents. LYL119 is an investigational ROR1-targeted CAR T-cell product that combines these technologies to create potent and durable CAR T cells. Healthy or NSCLC patient donor T cells were manufactured with the Epi-R protocols, activated with Stim-R or a standard reagent, and transduced with a vector encoding a ROR1 CAR and c-Jun. The NR4A3 gene or a control gene was edited using SpyFiTM Cas9 nuclease (Aldevron®). Cytotoxicity, cytokine production, phenotype, and single-cell transcriptomic and epigenetic profiles were evaluated in vitro after antigen restimulation assays designed to promote exhaustion. CAR T cell activity was evaluated in vivo using a ROR1+ NSCLC xenograft mouse model. Research and clinical scale LYL119 products achieved ~90% genome editing efficiency at the NR4A3 target gene resulting in a 13-fold protein reduction compared to non-edited CAR T cells. LYL119 exhibited superior cytotoxicity and cytokine production upon antigen restimulation across 7 different ROR1+ solid tumor cell lines compared to CAR T cells that lacked one or more reprogramming technologies. After repeated rounds of tumor cell killing, LYL119 displayed reduced surface expression of exhaustion-related receptors (e.g. TIM-3) and higher expression of stemness-related markers (e.g. CD127) compared to non-edited CAR T cells. Furthermore, transcriptomic analysis revealed global downregulation of exhaustion-related gene signatures and retention of unique cell subsets characterized by upregulation of memory and effector-associated gene signatures. LYL119 exhibited robust antitumor efficacy in vivo across a 10-fold dose range, including a very low dose of 1 × 105 CAR T cells. Lastly, LYL119 derived from NSCLC patient donor T cells also demonstrated enhanced cytotoxicity in vitro compared to control CAR T cells. These nonclinical data suggest LYL119, which combines c-Jun overexpression, NR4A3 KO, Epi-R protocols, and Stim-R technology, can limit exhaustion, maintain stem-like features, and has the potential to provide effective and durable CAR T-cell antitumor activity in patients with ROR1+ solid tumors. Citation Format: Viola C. Lam, Aileen Li, Meritxell Galindo Casas, Jessica Barragan, Christina Cheung, Jessica Briones, Esha Afreen, Grant Vavra, Jia Lu, Purnima Sundar, Rowena Martinez, Candace Sims, Shobha Potluri, Omar Ali, Alexander S. Cheung, Rachel C. Lynn. LYL119, an investigational ROR1-targeted CAR T-cell product incorporating four novel reprogramming technologies designed for effective cell therapy for solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 49.

Abstract 48: T cell-specific in vivo transduction with preclinical candidate KLN-1010 generates BCMA directed CAR T cells with potent anti-multiple myeloma activity

Abstract Introduction: Multiple Myeloma (MM) is the second most diagnosed hematologic malignancy and remains an incurable disease. KLN-1010 is a novel treatment for MM currently in preclinical development that uses our iGPSTM platform, an envelope-modified lentiviral vector particle engineered for specific and efficient in vivo gene delivery, to transduce T cells. T cells modified in vivo by KLN-1010 express a fully human anti-BCMA CAR following a single IV injection without the need for additional treatments or conditioning chemotherapy. Results: Human T cells exposed to KLN-1010 in vitro resulted in transduction and expansion of anti-BCMA CAR T cells in a concentration dependent manner. The resulting CAR T cells were highly avid and specific to BCMA-expressing cell lines. KLN-1010 treatment of preclinical mouse models resulted in T cell-specific CAR expression with no evidence of off-target modification including in the lung and liver, except phagocytes. A varied CAR T cell lineage composition was observed that included effector and memory CD4 and CD8 T cells. A single dose of KLN-1010 caused potent anti-tumor efficacy and complete tumor regression in a stringent model that requires high dose levels of ex vivo-cultured CAR T cells similar to those commercially available. When the anti-BCMA CAR molecules used in KLN-1010 are compared in preclinical models to other clinically relevant CARs, we observed superior tumor control by the KLN-1010 anti-BCMA CAR molecules. Clinical application of iGPS technology was modeled in non-human primates (NHP) using an NHP T cell-targeting particle that expresses an anti-CD20 CAR. CAR T cell activity, assessed by B cell depletion, and tolerability were evaluated without prior lymphodepleting chemotherapy across a 10x dose range. Rapid clearance of the iGPS particles from the blood was observed at all dose levels. Even at a low dose of 1.3e8 IU/kg, potent and durable CAR T cell activity was observed as evidenced by complete B cell depletion lasting over two months. Multiple CAR T cell expansions and contractions were observed during this period. The treatment at all dose levels was highly tolerable with no observed toxicities, cytokine release syndrome, or neurotoxicity. Conclusions: These data demonstrate that KLN-1010 is safe and efficacious in preclinical models and may offer greater accessibility than other therapeutic modalities to address an unmet medical need in MM. Without requirements of ex vivo manufacturing, lymphodepletion and inpatient treatment that restrict patient access to therapy, the iGPS technology is well poised to provide significant clinical benefit for a multitude of indications as an off-the-shelf, single dose treatment. Citation Format: Emily T. Beura, Howard J. Latimer, Denise Wong, Jenifer Obrigewitch, Alyson J. Warr, Shirley Dong, Matty Ariel, Josue Figueroa, Joseph Figueroa, Connor S. Dobson, Sanath K. Janaka, Alexander J. Najibi, Maggie Lau, Shannon Contrastano, Kevin Friedman. T cell-specific in vivo transduction with preclinical candidate KLN-1010 generates BCMA directed CAR T cells with potent anti-multiple myeloma activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 48.

Abstract 2054: A sensitive and specific human primary stem cell-based in vitro assay for predicting the gastrointestinal toxicity risk of therapeutic agents

Abstract Gastrointestinal (GI) toxicities are the most common drug-induced adverse events in human clinical trials. Drugs that cause GI toxicities often affect the proliferative stem and progenitor cell populations responsible for maintaining the cellular composition, self-renewing capacity, and barrier function of the intestinal epithelium. This disruption leads to impaired GI barrier integrity, culminating in a broad range of clinical symptoms, including diarrhea, inflammation, and increased risk of infection. The impact of GI toxicity is substantial and can cause dose-limiting clinical side-effects that may affect patient compliance and dosing adherence. Common nonclinical animal models, including rats, mice and dogs are often poor predictors of human GI toxicity and safety outcomes. Furthermore, in vivo animal testing poses limited screening throughput, which is particularly important in fields such as oncology, where matrices of double or triple drug combinations represent the standard of care. Additionally, existing cell-based model systems, such as Caco-2 cells, are of limited predictive value as they are tumor-derived and do not reprise native epithelium. Thus, there is a need for the development of assay systems that more accurately recapitulate human biology, have higher throughput testing capabilities, and ultimately improve clinical predictability. To this end, we have developed an assay to assess GI toxicity potential using the RepliGut Planar platform, which consists of a primary human stem cell-derived epithelium in a 96-well Transwell format. Quantitative dose-response curves were generated on proliferative and/or differentiated GI cultures for various test articles utilizing endpoints that assess GI proliferative capacity, cell viability, and epithelial barrier formation and maintenance. An initial set of marketed drugs (including diverse anti-proliferative, cytotoxic, or anti-inflammatory mechanisms) with known clinical incidence of diarrhea were over a 5-log dose range to enable modeling of the Toxic Concentration 50% (TC50). When benchmarked against the known human plasma concentrations (Cmax), this model was able to accurately predict clinical outcomes for diarrheagenic-drugs, as well as non-toxic negative controls, within 30-fold of the clinical Cmax. Overall, this model system provides straightforward and informative screening for GI toxicity that integrates sensitive and robust endpoints, enables screening to support early nonclinical development, and uniquely allows for mechanistic insights. Citation Format: James Levi, Reganne Lorichon, Lauren Boone, Maureen Bunger, Elizabeth Boazak, William R. Thelin. A sensitive and specific human primary stem cell-based in vitro assay for predicting the gastrointestinal toxicity risk of therapeutic agents [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2054.

First-in-human phase I study of BEBT-109 in previously treated EGFR exon 20 insertion-mutated advanced non-small cell lung cancer

BEBT-109 is an oral pan-mutant-selective inhibitor of epidermal growth factor receptor (EGFR) that demonstrated promising antitumor potency in preclinical models. This first-in-human study was a single-arm, open-label, two-stage study. Phase Ia dose-escalation study evaluated the safety and pharmacokinetics of BEBT-109 in 11 patients with EGFR T790M-mutated advanced non-small cell lung cancer (aNSCLC). Phase Ib dose-expansion study evaluated the safety and efficacy of BEBT-109 in 18 patients with EGFR exon 20 insertion (ex20ins)-mutated treatment-refractory aNSCLC. The primary outcomes were adverse events and antitumor activity. Clinical trial registration number CTR20192575. The phase Ia study demonstrated no dose-limiting toxicity, no observation of the maximum tolerated dose, and no new safety signals with BEBT-109 in the dose range of 20-180mg/d, suggesting that BEBT-109 had an acceptable safety profile among patients with EGFR T790M-mutated aNSCLC. Plasma pharmacokinetics of BEBT-109 showed a dose-proportional increase in the area under the curve and maximal concentration, with no significant drug accumulation. The dose-expansion study demonstrated that BEBT-109 treatment was tolerable across the three dose levels. The three most common treatment-related adverse events were diarrhea (100%; 22.2% ≥Grade 3), rash (66.7%; 5.6% ≥Grade 3), and anemia (61.1%; 0% ≥Grade 3). The objective response rate was 44.4% (8 of 18). Median progression-free survival was 8.0months (95% confidence intervals, 1.33-14.67). Preliminary findings showed that BEBT-109 had an acceptable safety profile and favorable antitumor activity in patients with refractory EGFR ex20ins-mutated aNSCLC. National Natural Science Foundation of China.

Abstract 5111: Rational synergistic radiosensitization strategies for group 3 medulloblastoma

Abstract Medulloblastoma (MB) is the most common malignant pediatric brain tumor. MB is comprised of 4 groups, termed Wnt, Shh, Group 3 and Group 4, each characterized by distinct molecular and clinical profiles. MYC-amplified Group 3 MB is associated imminent fatal metastatic relapse following standard treatment approaches, which currently include surgery, craniospinal irradiation and chemotherapy, highlighting an urgent need for identification of therapeutic vulnerabilities for these tumours. As such, using 4 novel patient-derived models of Group 3 MB that faithfully recapitulate the molecular and histological tumor characteristics, we characterised response to radiation of Group 3 MB in vitro to show that these tumors do not possess intrinsic radiation resistance. Furthermore, cumulative radiation experiments do not increase bulk radiation resistance in MB cells, highlighting the contribution of dynamic cell states to radiation response. In contrast, exposure to radiation leads to changes in cell clonogenic capacity as measured by limited dilution assays. Furthermore, RNA-seq data uncovered altered transcriptomic programs in response to cumulative radiation treatment. In particular, we observe differential regulation of genes encoding potassium ion channels and their regulators as well as genes encoding ECM and cell adhesion proteins. As part of our goal to evaluate the current multimodal treatment strategy of MB, we also show that a set of standard chemotherapeutic agents (etoposide, thiotepa and lomustine) currently used in the clinic do not produce a potent response at optimal doses in vitro, potentially accounting for abysmal survival outcomes seen in chemotherapy treated MB patients. As such, we conducted a screen of 144 FDA approved cancer drugs and identified therapeutics that selectively target Group 3 MB cells in contrast to normal tissue controls (foetal neural stem cells) in the low nanomolar dose range. In particular, topoisomerase inhibitor topotecan produces a potent response specific to MYC-amplified Group 3 MB, and sensitizes 2 patient-derived MB models to gamma irradiation. Altogether, our data characterizes Group 3 MB molecular and cellular responses to current treatment modalities and provides rationale for new clinical strategies to sensitize patients to radiation that will potentially allow to reduce the required radiation doses to treat these aggressive tumors, reduce the incidence of radiation treatment-related morbidity as well as improve cure rates. Citation Format: Julija Povilaikaite, Kaitlin Stitz, Nikhita Austin, Vijay Ramaswamy. Rational synergistic radiosensitization strategies for group 3 medulloblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5111.

Abstract 4649: BLX-3030, a potent, selective, orally available small molecule CDK9 inhibitor for aggressive variant prostate cancers

Abstract Background: The use of novel, potent targeted therapies in prostate cancers has led to the development of highly aggressive variant cancers, including neuroendocrine prostate cancer (NEPC). Gene amplification of N-MYC and C-MYC oncogenes leads to its overexpression in NEPC and mCRPC patients, highly prevalent in these lethal subtypes of cancers detected in 2% of all Prostate Cancers (PCa) and over 10-17% of mCRPC patients. N-MYC is a transcriptional target of CDK9, which acts as an oncogenic driver sufficient to transform human-derived prostate cancer to take on NEPC phenotypic changes resulting in a more aggressive disease identified in late-stage human PCa patients. Additionally, N-MYC and C-MYC are required for tumor drug resistance, and their downregulation through CDK9 inhibition lowers tumor growth. Our efforts have demonstrated that targeting both N-MYC and C-MYC as a driver of NEPC and mCRPC with highly selective CDK9 inhibitor BLX-3030 is a viable approach for therapeutic intervention of mCRPC and NEPC. Materials and Methods: Our empirical data-driven AI/ML MolecuLernTM fragment library, workflows, LigEdit design strategies, and hit2-lead optimization methods are employed. In vitro experiments include kinase glo, cell titer glo, selectivity profiler, ADME-Tox, FACS/apoptosis, cell migration, live cell imaging, immunofluorescent, western blot, combination studies and in vivo experiments including PK, prostate tumor models, 7-day tox, BLX-3030 non (GMP) scaleup and safety pharmacology experiments conducted. Results: We synthesized a series of novel small molecule CDK9 inhibitors that reversibly bind to CDK9, competitively block the ATP site with an IC50 of 1.06 nM, and elicit pharmacological responses in N-MYC, C-MYC, and other prostate cancer cells in vitro and in vivo. We studied the CDK9 N-MYC expression detected in PC-3, DU-145, LNCaP, and in LASCPC-01, NCI-H660, and 22RV1 cell lines. BLX-3030 exhibited potent cellular efficacy with an IC50 of 22 to 590 nM in PC cell lines and in N-MYC expressed cells had an activity of 76, 470, and 132 nM respectively, and reduced pCDK9, C-MYC, Mcl-1, N-MYC and its downstream RNA Pol II (Ser-2) and pAR (Ser-81) dose-dependently. BLX-3030 exhibits in vivo antitumor activity in LASCPC-01, 22RV1, and C4-2 prostate cancer mouse models. Several ADME-Tox, MTD, dose range finding, hERG and other in vivo safety pharmacology experiments have advanced BLX-3030 to the IND enabling studies. Conclusions: BLX-3030 is currently being investigated in non-clinical toxicology, toxicokinetic, and core battery of safety pharmacology studies including the assessment of effects on cardiovascular, central nervous, and respiratory systems. Citation Format: Zhaoang li, Chenyu Lin, Kyle Medley, Hariprasad Vankayalapati, David J. Bearss. BLX-3030, a potent, selective, orally available small molecule CDK9 inhibitor for aggressive variant prostate cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4649.

Abstract 3912: ATM and PARP combined inhibition demonstrate synergistic antitumor efficacy in osteosarcoma models

Abstract Signatures of BRCAness are found in many osteosarcoma (OS) tumors, driving an interest in PARP inhibition (PARPi) for OS treatment. Although PARPi have shown limited efficacy as OS monotherapy, rational combination strategies have not yet been explored for this disease. We performed a genome-scale loss-of-function CRISPR-Cas9 screen in two OS cell lines in the presence and absence of olaparib, a small molecule PARPi. ATM knockout (KO) scored highly as a potent synergizer of PARPi across both cell lines. Here we report this screen finding as well as in vitro validation of PARPi and ATM inhibition (ATMi) synergy in OS models. OS cell lines SAOS2 and CAL72 were infected with the Avana genome-scale lentiviral CRISPR library, targeting 20,000 genes with 4 uniquely barcoded guides (sgRNAs) per gene. After infection and antibiotic selection, cells were grown in an IC40 dose of olaparib or matched control for 18 days. Following DNA sequencing of barcodes and rigorous quality control, sgRNA dropout hits were calculated against the control arm. ATM KO sensitizing to PARPi was first validated utilizing a CRISPR-based genetic approach. ATM KO in CAL72 and SAOS2 was performed via lentiviral infection. After KO confirmation by western immunoblotting (WB), ATM KO and control KO cells were treated with PARPi in a dose range of 19.5 nM-10 µM. ATP-based and Incucyte assays were used to quantify differential cell viability. Secondly, the combination of a small molecule ATMi and a PARPi was studied in a panel of OS cell lines. Synergistic cytotoxicity was quantified using the Zero Interaction Potency (ZIP) model. DNA damage levels after PARPi and ATMi (by chemical inhibition and genetic KO) were assessed by WB for γH2AX, a marker of DNA double-stranded breaks (DSBs). In our genome-scale CRISPR screens, ATM arose as a top druggable hit across both lines. Reassuringly, PARP1 KO scored as a top resistance mediator to PARPi, confirming biologic relevance of the screen and supporting the known PARP-trapping mechanism of these drugs in OS. At low-throughput, ATM KO lines trended toward increased sensitivity to PARPi in short-term viability assays. Combined chemical inhibition yielded highly synergistic ZIP scores across OS cell lines; synergy was achieved with both drugs in the low nanomolar range. γH2AX levels were substantially increased in the setting of combined PARPi and ATMi (via chemical inhibition or genetic KO) as compared to inactivation of either PARP or ATM alone, indicating increased presence of DNA DSBs. In conclusion, these data demonstrate synergy of ATM and PARP inhibition, providing promise for their potential combined use in the treatment of OS. Accumulation of DNA DSBs in response to PARP and ATM inactivation suggests a shared role in DNA damage repair as the molecular basis of synergism. Ongoing studies are investigating anti-tumor potential in in vivo OS models and exploring the mechanistic impact of the combination in OS tumors. Citation Format: Sona N. Kocinsky, Janeala J. Morsby, William C. Wright, Monika Wierdl, Caroline S. Wechsler, Gabriela Alexe, Paul Geeleher, Kimberly Stegmaier, Lillian M. Guenther. ATM and PARP combined inhibition demonstrate synergistic antitumor efficacy in osteosarcoma models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3912.

Monitoring of changes in the concentration of volatile organic compound in beef irradiated with accelerated electrons

The need to develop safe methods for radiation processing of food products to improve their quality and extend their shelf life stimulates new scientific research aimed at increasing their effectiveness. Oxidation of lipids and proteins occurred under the impact of ionizing radiation in products with a high fat and water content, such as chilled meat and fish products, leads to the formation of volatile organic compounds in the product thus giving it a specific smell and taste. During storage, biochemical processes associated with microbial enzymatic activity and auto-oxidation develop in processed refrigerated products. These processes also modify the volatile organic compounds, which affect the organoleptic properties of the product. The method of gas chromatography-mass spectrometry was used to study the behavior of volatile compounds identified in irradiated beef samples both immediately after irradiation and four days later to determine the effective dose range for the radiation processing of beef. Monitoring of the content of volatile compounds in beef samples irradiated by 1-MeV electrons within a dose range from 0.25 to 5 kGy showed that the content of certain alcohols, aldehydes, and alkanes exhibited pronounced dose- and time-dependent character. The developed mathematical model describes the dependence of the concentration of volatile compounds identified immediately after irradiation in beef samples on the irradiation dose. The model is based on the simultaneous occurrence of two competing processes: the decomposition of compounds due to their oxidation and accumulation due to oxidation of other compounds after exposure to ionizing radiation. The results obtained revealed that the effective dosage range of radiation treatment lies between 250 and 1000 Gy.

First-in-Human Phase 1 Study Evaluating the Safety, Pharmacokinetics, and Pharmacodynamics of DISC-0974, an Anti-Hemojuvelin Antibody, in Healthy Participants.

Pathologic elevations in hepcidin, a key regulator of iron homeostasis, contribute to anemia of inflammation in chronic disease. DISC-0974 is a monoclonal antibody that binds to hemojuvelin and blocks bone morphogenetic protein signaling, thereby suppressing hepcidin production. Reduction of systemic hepcidin levels is predicted to increase iron absorption and mobilize stored iron into circulation, where it may be utilized by red blood cell (RBC) precursors in the bone marrow to improve hemoglobin levels and to potentially alleviate anemia of inflammation. We conducted a first-in-human, double-blind, placebo-controlled, single-ascending dose study to evaluate safety, pharmacokinetics, and pharmacodynamics of DISC-0974 in healthy participants. Overall, 42 participants were enrolled and received a single dose of placebo or DISC-0974 at escalating dose levels (7-56mg), administered intravenously (IV) or subcutaneously (SC). DISC-0974 was well tolerated, with a safety profile comparable to that of placebo. Pharmacokinetic data was dose and route related, with a terminal half-life of approximately 7 days. The bioavailability of SC dosing was ∼50%. Pharmacodynamic data showed dose-dependent decreases in serum hepcidin, with reductions of nearly 75% relative to baseline at the highest dose level tested, and corresponding increases in serum iron in response to DISC-0974 administration. Dose-dependent changes in serum ferritin and hematology parameters were also observed, indicating mobilization of iron stores and downstream effects of enhanced hemoglobinization and production of RBCs. Altogether, these data are consistent with the mechanism of action of DISC-0974 and support the selection of a biologically active dose range for evaluation in clinical trials for individuals with anemia of inflammation.

Beta irradiation-induced thermoluminescence: Glow curve analysis and kinetic parameters in combustion-synthesized undoped Ca4YO(BO3)3

This study examines the thermoluminescent (TL) properties of undoped Ca4YO(BO3)3 phosphor, focusing on how it behaves under a variety of experimental conditions. The IRSL-TL 565 nm was chosen as the appropriate detection filter among various optical detection filter combinations. During the preheating trials conducted at a rate of 2 °C/s, the TL peak exhibited increased intensity, particularly around 200 °C. The experimental outcomes demonstrated a reliable linear relationship (R2 = 0.996 and b = 1.015) in the dose response of undoped preheated Ca4YO(BO3)3 within the range of 1–200 Gy. The investigation encompasses a range of techniques, including the TM-Tstop method, computerized glow curve deconvolution (CGCD) analysis, and theoretical modelling. The application of the TM-Tstop method to samples irradiated with a 5 Gy dose revealed distinct zones on the TM versus Tstop diagram, signifying the presence of at least two discernible components within the TL glow curve, specifically, a single general order kinetics peak and a continuous distribution. The analysis of activation energy versus preheated temperature exhibited a stepwise curve, indicating five trap levels with depths ranging between 1.13 eV and 1.40 eV. The CGCD method also revealed the superposition of at least five distinct TL glow peaks. It was observed that their activation energies were consistent with the Tm-Tstop experiment. Furthermore, the low Figure of Merit (FOM) value of 1.18% indicates high reliability in the goodness-of-fit measure. These findings affirm the reliability and effectiveness of the employed methods in characterizing the TL properties of the Ca4YO(BO3)3 phosphor under investigation. Theoretical models, including the semi-localized transition model, were introduced to explain anomalous observations in TL glow peak intensities and heating rate patterns. While providing a conceptual framework, these models may require adjustments to accurately capture the specific characteristics uncovered through CGCD analysis. As a potential application, the study suggests that the characterized TL properties of Ca4YO(BO3)3 phosphor could be utilized in dosimetric applications, such as radiation dose measurements, owing to its reliable linear response within a broad dose range.

Nonlinear Pharmacokinetics of Topical Flurbiprofen Gel in a Phase I Study Among Chinese Healthy Adults.

PDX-02 (Flurbiprofen sodium) is a topical nonsteroidal anti-inflammatory drug in gel formulation for local analgesia and anti-inflammation. A Phase I clinical trial was conducted to assess the safety, tolerability, and pharmacokinetics of single and multiple doses of PDX-02 gel in Chinese healthy adults. The trial comprised three parts: (1) a single-dose ascending study with three dose levels (0.5%, 1% to 2% PDX-02 gel) applied on a 136 cm2 skin area; (2) a multiple-dose study with either 1% or 2% PDX-02 gel applied on a 136 cm2 skin area for 7 consecutive days; and (3) a high dose group with 2% PDX-02 gel on an 816 cm2 skin area and a frequent multiple dose group with 2% PDX-02 gel on a 272 cm2 skin area four times a day for 7 consecutive days. The safety, tolerability and pharmacokinetics of the PDX-02 gel were evaluated in each part. A total of sixty participants completed the trial, with all adverse events recovered and all positive skin reaction being transient and recovered. The overall absorption of topical PDX-02 gel was slow with a mean peak time exceeding 9h. The elimination rate remained consistent between dose groups. A less-than-dose-proportional nonlinear pharmacokinetics relationship was observed within the studied dose range, and this is likely due to the autoinduction of skin first-pass metabolism. The topical PDX-02 gel showed favorable safety and tolerability in both single and multiple dosing studies, with a less-than-dose-proportional nonlinear pharmacokinetics observed.

Population pharmacokinetics of a novel PCSK9 antisense oligonucleotide.

The aim of this study was to characterize the population pharmacokinetics of AZD8233, an antisense oligonucleotide (ASO) that targets the PCSK9 transcript to reduce hepatocyte PCSK9 protein production and plasma levels. AZD8233 utilizes generation 2.5S-constrained ethyl motif (cET) chemistry and is conjugated to a triantennary N-acetylgalactosamine (GalNAc3) ligand for targeted hepatocyte uptake. A non-linear mixed-effect modelling approach utilizing NONMEM software was applied to AZD8233 concentration-time data from 3416 samples in 219 participants from four phase 1-2 studies, one in healthy volunteers (NCT03593785) and three in patients with dyslipidaemia (NCT04155645, NCT04641299 and NCT04823611). The final model described the AZD8233 plasma concentration-time profile from four phase 1-2 studies in healthy volunteers or participants with dyslipidaemia, covering a dose range of 4 to 120 mg. The pharmacokinetics of AZD8233 were adequately described by a two-compartment model with first-order absorption. The supra-proportional increase in maximum plasma concentration (Cmax) across the observed dose range was described by non-linear Michaelis-Menten elimination (maximum elimination rate, 9.9mg/h [12% relative standard error]; concentration yielding half-maximal elimination rate, 4.8mg/L [18% relative standard error]). Body weight, sex, estimated glomerular filtration rate and disease status (healthy participant vs. patient with dyslipidaemia) were identified as factors affecting exposure to AZD8233. Covariate analysis showed body weight to be the main factor affecting exposure to AZD8233, which largely explained the higher Cmax observed in the Asian population relative to non-Asians.

Ferroptosis inhibitors reduce celastrol toxicity and preserve its insulin sensitizing effects in insulin resistant HepG2 cells

ObjectiveResearch has shown that celastrol can effectively treat a variety of diseases, yet when passing a certain dosage threshold, celastrol becomes toxic, causing complications such as liver and kidney damage and erythrocytopenia, among others. With this dichotomy in mind, it is extremely important to find ways to preserve celastrol’s efficacy while reducing or preventing its toxicity. MethodsIn this study, insulin-resistant HepG2 (IR-HepG2) cells were prepared using palmitic acid and used for in vitro experiments. IR-HepG2 cells were treated with celastrol alone or in combination with N-acetylcysteine (NAC) or ferrostatin-1 (Fer-1) for 12, 24 or 48 h, at a range of doses. Cell counting kit-8 assay, Western blotting, quantitative reverse transcription-polymerase chain reaction, glucose consumption assessment, and flow cytometry were performed to measure celastrol’s cytotoxicity and whether the cell death was linked to ferroptosis. ResultsCelastrol treatment increased lipid oxidation and decreased expression of anti-ferroptosis proteins in IR-HepG2 cells. Celastrol downregulated glutathione peroxidase 4 (GPX4) mRNA. Molecular docking models predicted that solute carrier family 7 member 11 (SLC7A11) and GPX4 were covalently bound by celastrol. Importantly, we found for the first time that the application of ferroptosis inhibitors (especially NAC) was able to reduce celastrol’s toxicity while preserving its ability to improve insulin sensitivity in IR-HepG2 cells. ConclusionOne potential mechanism of celastrol’s cytotoxicity is the induction of ferroptosis, which can be alleviated by treatment with ferroptosis inhibitors. These findings provide a new strategy to block celastrol’s toxicity while preserving its therapeutic effects.Please cite this article as: Liu JJ, Zhang X, Qi MM, Chi YB, Cai BL, Peng B, Zhang DH. Ferroptosis inhibitors reduce celastrol toxicity and preserve its insulin sensitizing effects in insulin resistant HepG2 cells. J Integr Med. 2024; 22(3): 286–294.

Biochar-enhanced anaerobic mixed culture for biodegradation of 1,2-dichloroethane: Microbial community, mechanisms, and techno-economics

While anaerobic digestion (AD) has been employed for the degradation of chlorinated aliphatic hydrocarbons, the associated digester performance might suffer from volatile fatty acids accumulation, insufficient substrate-microbes interaction, and lower biogas yields. To overcome these limitations, this study is the first to augment the hydrocarbon-degrading microbial capacities by adding agricultural waste-based biochar to the digestion medium. 1,2-dichloroethane (1,2-DCA) was selected as the target pollutant because it is discharged in large quantities from oil refining, petrochemical, and chemical industries, causing serious environmental and human health concerns. A multi-chamber anaerobic reactor (MAR) was operated at a 1,2-DCA loading rate of 1.13 g/L/d, glucose dosage (as an electron donor) range of 200–700 mg/L, and hydraulic retention time of 11.2 h, giving dechlorination = 32.2 ± 6.9% and biogas yield = 210 ± 30 mL/g CODremoved. These values increased after biochar supplementation (100 mg/g volatile solids, VS, as an inoculum carrier) up to 60.2 ± 11.5% and 290 ± 40 mL/g CODremoved, respectively, owing to the enhancement of dehydrogenase enzyme activities. Burkholderiales (15.3%), Clostridiales (2.3%), Bacteroidales (3.5%), Xanthomonadales (3.3%), and Rhodobacterales (6.1%) involved in 1,2-DCA degradation were dominant in the reactor supplemented with biochar. It's suggested that biochar played a major role in facilitating the direct interspecies electron transfer (DIET) between syntrophic bacteria and methanogens, where chloride, ethylene glycol, and acetate derived from 1,2-DCA dechlorination could be further used to promote methanogenesis and methane production. The synergetic effect of adsorption and dechlorination towards 1,2-DCA removal was validated at various biochar dosages (50–120 mg/g) and 1,2-DCA concentrations (50–1000 mg/L). The techno-economic results showed that the cost of treating 1,2-DCA-laden discharge (100 m3/d) by the MAR module could be 0.83 USD/m3 with a payback period of 6.24 years (NPV = 2840 USD and IRR = 10%), retrieving profits from pollution reduction (9542 USD/yr), biogas selling (10418 USD/yr), and carbon credit (10294 USD/yr).

Hygienic efficiency of ultraviolet disinfection of water in centralized drinking and household water supply systems (systematic review)

Introduction. Drinking water supplied to the population must be safe in epidemic terms. However, some viruses and parasites are resistant to the disinfecting effect of traditionally used agents for water treatment. The use of UV irradiation ensures the microbiological safety of water, but there are factors affecting its effectiveness. 
 The purpose of the review was to systematize scientific data on the effectiveness of the use of UV irradiation in relation to drinking water.
 Materials and methods. The search for scientific publications was carried out using literature databases MedLine/PubMed, Scopus, and Science Direct. The total number of publications was one thousand six hundred forty-six. The criteria for including the report in the systematic review were: publications in Russian or English with an available full-text version; samples for the study are water from centralized water supply systems or specially prepared microorganisms; any type of research evaluating the effectiveness of a wide range of doses of UV irradiation against microorganisms.
 Results of the analysis of publications. A systematic review included 17 reports about the effect of UV irradiation on bacteria and viruses. The subject of several studies was the resistance of protozoan cysts and helminth eggs to different doses of UV irradiation. According to the results presented in the publication, pathogenic viruses and bacteria, giardia cysts possess of the least resistance to UV irradiation (100% inactivation). 4 log-inactivation was established for Mycobacteia. E. coli and coliphages demonstrate a wide range of indicators of the resistance to ultraviolet radiation due to the diversity of the studied strains. 
 Conclusion. UV disinfection should be used as an additional method in combination with traditional reagents methods of water treatment. It is necessary to study the effect of UV irradiation on helminth eggs and protozoan cysts.

Low-dose targeted radionuclide therapy synergizes with CAR T cells and enhances tumor response.

Ionizing radiation has garnered considerable attention as a combination partner for immunotherapy due to its potential immunostimulatory effects. In contrast to the more commonly used external beam radiation, we explored the feasibility of combining chimeric antigen receptor (CAR) T cell therapy with targeted radionuclide therapy (TRT), which is achieved by delivering β-emitting 177Lu-DOTATATE to tumor via tumor-infiltrating CAR T cells that express somatostatin receptor 2 (SSTR2). We hypothesized that the delivery of radiation to tumors could synergize with CAR T therapy, resulting in enhanced antitumor immunity and tumor response. To determine the optimal dosage and timing of 177Lu-DOTATATE treatment, we measured CAR T cell infiltration and expansion in tumors longitudinally through positron emission tomography (PET) using a SSTR2-specific positron-emitting radiotracer,18F-NOTA-Octreotide. In animals receiving CAR T cells and a low-dose (2.5 Gy) of TRT following the administration of 177Lu-DOTATATE, we observed a rapid regression of large subcutaneous tumors, which coincided with a dramatic increase in serum proinflammatory cytokines. Tumor burden was also reduced when a higher radiation dose (6 Gy) was delivered to the tumor. However, this higher dose led to cell death in both the tumor and CAR T cells. Our study suggests that there may exist an optimum range of TRT dosage that can enhance T cell activity and sensitize tumor cells to T cell killing, which may result in more durable tumor control compared to a higher radiation dose.