Fulcrum Therapeutics (United States)

ABSTRACTChimeric antigen receptor T (CAR‐T) cells have been used to treat patients with glioblastoma (GBM) in clinical trial settings by targeting GBM‐associated antigens. However, the efficacy of these CAR‐T cells remains limited mainly due to the heterogeneous expression of tumor antigen and their anergy in the tumor microenvironment (TME). Cytokine‐inducible SH2‐containing protein (CIS, encoded by the gene CISH) is a potent intracellular checkpoint inducing T‐cell anergy. Here, we identified fibroblast activation protein alpha (FAPα) as a highly attractive target for CAR‐T cell therapy against GBM based on its dual expression pattern (on tumor cells and perivascular cells) in GBM. A panel of nanobodies specific for FAPα was isolated, and FAPα‐targeting CAR‐T cells were developed using the isolated nanobody to verify their specific cytotoxicity to GBM cells. Furthermore, a non‐viral circular single‐stranded DNA (cssDNA)‐based CRISPR/Cas9‐targeted genome‐editing (cssDNA/CRISPR/Cas9) technology was used to integrate CAR cassettes at the CISH locus to generate CISH‐knockout (CISH‐KO) CAR‐T cells. The resulting CISH‐KO‐CAR‐T cells exhibited robust proliferation and potent anti‐GBM activity in vitro and in vivo. Thus, our results provide novel engineered CAR‐T cells with enhanced efficacy against GBM.

Abstract Background Outpatients presenting with signs and symptoms of uncomplicated urinary tract infection (uUTI) are typically managed with an empiric course of antibiotic therapy. Treatment failure resulting in the need for repeated courses of antibiotics may occur in patients with certain baseline characteristics. In the absence of microbiologic data, typically not available to the prescribing clinician at the time of the clinical encounter, there is a need to define baseline variables predictive of treatment failure in patients with clinical evidence of uUTI. We recently completed two phase 3 studies, SURE-1 and REASSURE, evaluating 3,874 adult women with uUTI. Association Between Baseline Variables and Treatment Outcome Methods SURE-1 and REASSURE were randomized, double-blinded, double-dummy, multicenter Phase 3 clinical trials. In the SURE-1 trial, patients with uUTI were randomized to five days of oral sulopenem or three days of ciprofloxacin. In the REASSURE trial, patients with uUTI were randomized to five days of oral sulopenem or five days of amoxicillin/clavulanate. For both trials, the primary endpoint was overall success, defined as both clinical and microbiologic response, at the Day 12/Test of Cure (TOC) visit. Post hoc analyses were performed to determine if there was a relationship between baseline characteristics and overall success for these clinical trial patients. Results In the SURE-1 and REASSURE trials combined, there were 2,061 adult female patients with uUTI in the microbiologic Modified Intent to Treat (mMITT) population. The majority of baseline uropathogens recovered were Escherichia coli (83.4%) and Klebsiella pneumoniae (10.0%). Overall response could be determined for 1,958 (95%) of the patients in the mMITT population. For these clinical trial patients, age > 65 years, BMI > 30 kg/m2, creatinine clearance < 60 mL/min and Diabetes mellitus were associated with treatment failure (Table). Conclusion Older age, higher BMI, lower creatinine clearance and the presence of Diabetes mellitus identified uUTI patients at increased risk for treatment failure. These variables, plus infection known or suspected to be resistant to one or more commonly prescribed oral antibiotic, may serve to guide clinicians when choosing empiric antibiotic therapy for adult women with uUTI. Disclosures Steven I. Aronin, MD, Iterum Therapeutics: Employee|Iterum Therapeutics: Stocks/Bonds (Public Company) Sailaja Puttagunta, MD, Iterum Therapeutics: Stocks/Bonds (Public Company) Michael Dunne, MD, Iterum Therapeutics: Board Member|Iterum Therapeutics: Employee|Iterum Therapeutics: Stocks/Bonds (Public Company)

Background T cell engagers (TCEs) are powerful therapeutics which can kill cells expressing low levels of target antigen.To limit on-target off-tumor toxicity, it is critical to identify antigens with limited normal tissue expression or apply strategies for conditional engagement.While few antigens are completely tumor-specific, tumor cells may co-express pairs of antigens which are not co-expressed on normal cells.By selectively targeting such antigen pairs but not the individual antigens, AND-gated TCEs can impart anti-tumor efficacy with reduced toxicity.Here, we aimed to identify and validate a tumor-specific antigen pair for a potential AND-gated TCE.Methods Gene signatures were used to identify tumor cells in publicly available single cell RNA-sequencing (scRNA-seq) datasets for colorectal (CRC), 1 2 non-small cell lung (NSCLC), [3][4][5] and breast cancer (BC). 6 7Next, we compiled genes predicted to encode cell-surface proteins. 8To find coexpressed genes, Weighted Gene Correlation Network Analysis (WGCNA) was applied. 9were then analyzed for normal tissue expression using published scRNA-seq data. [10][11]1][12] For one tumor-specific pair, membrane co-expression was examined via multiplex immunohistochemistry (IHC) on normal and tumor tissue-derived microarrays (TMAs).In addition, five adenocarcinoma CRC tissue blocks with known mesothelin (MSLN) expression and five unbiasedly chosen mucinous CRC tissue blocks were also assessed.Results From 66 co-expressed tumor antigen pairs identified by WGCNA in more than one dataset, only three were tumor-specific.Target validation was pursued for mucin 13 (MUC13) and MSLN, due to expression of the pair in CRC and NSCLC and known toxicities of therapies targeting MSLN. [12][13]3][14][15] On a normal TMA, low levels of dual-positive staining for MUC13xMSLN were observed in 3 non-essential tissues.On tumor-derived TMAs, 37% (11/30) of CRC and 0% (0/30) of NSCLC samples were double-positive, consistent with the bioinformatics data where 18% (10/55) of CRC and 4% (2/50) of NSCLC patients showed co-expression.Doublepositive tissue blocks for adenocarcinoma (4/5) and mucinous CRC (3/5) showed respective fractions of double-positive cells of 1-15% and 1-50%.Conclusions To our knowledge, this is the first report of MUC13xMSLN as a potential tumor-specific antigen-pair in CRC.An AND-gated MUC13xMSLN TCE may reduce toxicities seen with MSLN therapies, although the percentage of patients expressing the target pair and the frequency of dualpositive cancer cells in these patients is relatively low.Overall, this data suggests the described bioinformatics and protein validation approach is suitable for identifying targets for ANDgated therapies in other cancer subtypes.

Abstract Background SCD is the most common inherited blood disorder caused by a mutation in the β - globin (HBB) gene, resulting in dysfunctional hemoglobin S (HbS) that polymerizes under hypoxic conditions with consequent characteristic and pathogenic dysmorphic erythrocytes. Increased expression of fetal hemoglobin (HbF) can counteract this process, ameliorating clinical manifestations and symptoms of SCD and reducing morbidity and mortality. Pociredir, a novel, oral HbF inducer in development for the treatment of SCD, targets the embryonic ectoderm development subunit of polycomb repressive complex 2, inhibiting trimethylation of lysine 27 on histone H3, decreasing BCL11A expression and increasing HbF. We report on an ongoing phase 1b, open-label, multicenter, international study evaluating the safety, PK, and PD of oral pociredir in patients with SCD (PIONEER; ClinicalTrials.gov ID: NCT05169580). Methods The study was approved by regulatory authorities and ethics committees of participating sites. All participants provided written informed consent prior to their inclusion. PIONEER will enroll up to 70 patients 18-65 years of age with SCD (genotypes: S/S, S/β0, S/β+, and S/C). Current key inclusion criteria are poor response or intolerance of hydroxy urea (HU) and severe sickle cell disease. Exclusion criteria include prior hematopoietic stem cell transplant, or gene therapies; receiving scheduled transfusions; having known chromosomal abnormality or genetic mutation that may put the patient at increased risk of myelodysplastic syndrome or acute myeloid leukemia. Pociredir will be administered orally once daily for 12 weeks. Patients will be followed post dosing for 4 weeks. Primary endpoints are safety and tolerability and PK of pociredir. Secondary endpoints are changes from baseline in HbF and other SCD relevant biomarkers. Before a significant study amendment, patients enrolled across 3 cohorts. Cohort 1 enrolled 10 adults who received pociredir at a dose of 6 mg once daily (QD) for 4 weeks, with an option to continue for 8 more weeks. Cohorts 2 and 3 enrolled 2 adults (2 mg QD for 12 weeks) and 4 adults (12 mg QD), respectively. Concurrent hydroxyurea (HU) was allowed for patients included in the analysis. Results As of data cut off, 16 patients with HbSS genotype have been included in the analysis. Their mean age was 30.3 years (range, 21-48), and most participants were female (69%). Baseline mean HbF was 9.3% (range, 3.2-19.9%) and 5 patients were on HU. Ten patients had 23 adverse events (AEs), with 8 AEs deemed possibly related to pociredir, including headache (n = 2), lip numbness, diarrhea, tinnitus, fatigue, drowsiness, and nausea. All related AEs were mild, nonserious, resolved quickly, and did not lead to drug discontinuation. Four AEs were vaso-occlusive crises (VOCs); 1 was a serious AE involving ACS, unrelated to the drug, in a nonadherent patient. Other VOCs were mild to moderate and managed as outpatients. No laboratory-related AEs, deaths, or discontinuations occurred. Maximum HbF increase from baseline was 9.8% points at 12 weeks (Cohort 1) and 10.0% points at 6 weeks (Cohort 3). Most patients showed improved hemolysis biomarkers. Conclusions Preliminary safety, PK, and efficacy results demonstrate a favorable safety profile, a dose-dependent increase in HbF, and a reduction in hemolysis, supporting further investigation. PIONEER is currently enrolling in Nigeria, South Africa, and the United States.

Abstract Objective: Multiple aggressive cancers of unmet need have alterations involving the insulin-like growth factor 1 receptor (IGF-1R) pathway, including gene fusions and/or high IGF-1R expression, that correlate with poor outcomes. In Ewing sarcoma (ES) and desmoplastic small round cell tumor (DSRCT), the gene fusion-produced oncogenic proteins upregulate the IGF signaling pathway. Although prior attempts at inhibiting IGF-1R with non-payload bearing naked monoclonal antibodies or small molecules produced some tumor responses, none of these agents were ultimately approved, potentially due to suboptimal potency and escape mechanisms via redundant signaling. LX-101 is a next-generation, targeted therapy directed to the IGF-1R, consisting of a proprietary IGF-1 variant coupled to a cytotoxic methotrexate payload. LX-101 was previously evaluated in Phase 1 trials of adult patients with advanced, pretreated cancers, where it was well-tolerated and demonstrated single agent activity. Neither a dose limiting toxicity nor a maximum tolerated dose was reached, leaving room for additional dose and schedule optimization. Herein, we investigated the in vitro and in vivo anti-tumor activity of the payload-bearing agent LX-101 in ES and DSRCT. Methods: ES and DSRCT cell lines were incubated with LX-101 (1 - 10, 000 nM) for 72 h. Cell viability was assessed by CellTiter-Glo; IC50s were calculated using GraphPad PRISM software. Pretreatment IGF-1R expression levels were analyzed by RNA-seq & Western blot. NOD-SCID mice bearing subcutaneous A-673 tumors were treated i.v. with vehicle or LX-101 (16 uEq/kg, twice a week, for 3 weeks). Tumor volume and body weight were measured 3 times a week. Results: LX-101 displayed anti-tumor activity against ES and DSRCT cell lines, including EWSR1::FLI1+ cell lines A-673 (IC50 = 693 nM), ES-8 (IC50 = 354 nM), and TC-71 (IC50 = 199 nM), as well as the EWSR1::WT1+ cell line BOD-DSRCT (IC50 = 453 nM). IGF-1R mRNA levels correlated with LX-101 anti-tumor activity. In mice bearing A-673 tumors, LX-101 significantly inhibited tumor volume, in vivo, following twice a week treatment for 3 weeks (55% tumor growth inhibition relative to vehicle group on Day 21, p < 0.01). No adverse effects were observed, demonstrating that LX-101 was well-tolerated. Conclusion: These results demonstrate that LX-101 has potent anti-tumor activity against ES, in vivo and in vitro, and DSRCT, in vitro, providing a differentiated approach from previous IGF-1R targeting agents. These data support further clinical development of LX-101 in ES, DSRCT, and other IGF-1R-involved cancers. Phase 2 clinical trials are expected to begin in 2025. Citation Format: Matt Hoberman, Danh Dinh Truong, Roberto Cardenas Zuniga, Joseph A. Ludwig. LX-101, a novel, clinical stage, payload-bearing targeted therapy directed to the insulin-like growth factor receptor (IGF-1R), demonstrates potent anti-tumor activity in Ewing sarcoma animal models and desmoplastic small round cell tumor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 2929.