HEK293/Human 4-1BB / TNFRSF9 Stable Cell Line

Abstract: Lack of sustained response to oncogenic Kras (Kras*) inhibition in preclinical models and patients with pancreatic ductal adenocarcinoma (PDAC) emphasizes the need to identify impactful synergistic combination therapies to achieve robust clinical benefit. Kras* targeting results in an influx of global T cell infiltrates including Tregs, effector CD8 + T cells and exhausted CD8 + T cells expressing several immune checkpoint molecules in PDAC. Here, we probe whether the T cell influx induced by diverse Kras* inhibitors open a therapeutic window to target the adaptive immune response in PDAC. We show a specific synergy of anti-CTLA4 immune checkpoint blockade with Kras* targeting primed by Kras G12D allele specific inhibitor, MRTX1133 and multi-selective pan-RAS inhibitor, RMC-6236, both currently in clinical testing phase. In contrast, attempted therapeutic combination following Kras* targeting with multiple checkpoint inhibitors, including anti-PD1, anti-Tim3, anti-Lag3, anti-Vista and anti-4-1BB agonist antibody failed due to compensatory mechanisms mediated by other checkpoints on exhausted CD8 + T cells. Anti-CTLA4 therapy in Kras* targeted PDAC transcriptionally reprograms effector T regs to a naïve phenotype, reverses CD8 + T cell exhaustion and is associated with recruitment of tertiary lymphoid structures (TLS) containing interferon (IFN)-stimulated/ activated B cells and germinal center B cells to enable immunotherapy efficacy and overcome resistance with long-term survival. Single cell ATAC sequencing analysis revealed that transcriptional reprogramming of Tregs is epigenetically regulated by downregulation of AP-1 family of transcription factors including Fos, Fos-b, Jun-b, Jun-d in the IL-35 promoter region. This study reveals an actionable vulnerability in the adaptive immune response in Kras* targeted PDAC with important clinical implications.

Abstract: CD19-CAR-T-cells emerge as a major therapeutic option for relapsed/refractory B-cell-derived malignancies, however approximately half of patients eventually relapse. To identify resistance-driving factors, we repeatedly exposed B-cell lymphoma/B-cell acute lymphoblastic leukemia to 4-1BB/CD28-based CD19-CAR-T-cells in vitro . Generated models revealed costimulatory domain-dependent differences in CD19 loss. While CD19-4-1BB-CAR-T-cells induced combination epitope/total CD19 protein loss, CD19-CD28-CAR-T-cells did not drive antigen-escape. Consistent with observations in patients relapsing after CD19-4-1BB-CAR-T-cells, we identified CD19 frameshift/missense mutations affecting residues critical for FMC63 epitope recognition. Mathematical simulations revealed that differences between CD19-4-1BB- and CD19-CD28-CAR-T-cells activity against low-antigen-expressing tumor contribute to heterogeneous therapeutic responses. By integrating in vitro and in silico data, we propose a biological scenario where CD19-4-1BB-CAR-T-cells fail to eliminate low-antigen tumor cells, fostering CAR-resistance. These findings offer mechanistic insight into the observed clinical differences between axi-cel (CD28-based) and tisa-cel (4-1BB-based)-treated B-cell lymphoma patients and advance our understanding on CAR-T resistance. Furthermore, we underscore the need for specific FMC63 epitope detection to deliver information on antigen levels accessible for CD19-CAR-T-cells.

Abstract: In low-income and middle-income counties (LMICs), the outcome of relapsed or refractory B-cell malignancies is poor due to the absence of effective therapies. We report the results of a phase 1/2 study of a novel humanised anti-CD19 4-1BB chimeric antigen receptor (CAR) T-cell therapy, talicabtagene autoleucel, for patients with relapsed or refractory B-cell malignancies.This open-label, multicentre, phase 1/2 study was done at six tertiary cancer centres in India. Phase 1 was a single-centre study done in Tata Memorial Hospital, India, in patients aged 18 years or older with relapsed or refractory B-cell lymphomas. Phase 2 was a single-arm, multicentre, basket trial done in five tertiary cancer centres in patients aged 15 years and older with relapsed or refractory B-cell acute lymphoblastic leukaemia or B-cell lymphoma. Eligible patients had a life expectancy of 12 weeks or more, an ECOG performance status of 0-1 (phase 1) or 0-2 (phase 2), and an adequate organ function. Patients underwent apheresis to obtain at least 1 × 109 lymphocytes to manufacture CAR T cells. Lymphodepletion therapy was done with cyclophosphamide 500 mg/m2 and fludarabine 30 mg/m2 for 3 days or bendamustine 90 mg/m2 for 2 days. Patients were then infused intravenously with talicabtagene autoleucel 1 × 107-5 × 109 CAR T cells in a fractionated schedule (10%, 30%, and 60%, on days 0, 1, and 2, respectively) during phase 1 or at least 5 × 106 CAR T cells per kg (up to 2 × 109 CAR T cells) on day 0 during phase 2. The primary endpoints were safety (phase 1) and overall response rate (phase 2). The efficacy analysis was done in the efficacy evaluable cohort (all patients who received the target dose and 3 days of lymphodepletion therapy). The safety analysis was done in the safety population (all patients who received talicabtagene autoleucel). The trials are registered with Clinical Trial Registry-India (CTRI/2021/04/032727 and CTRI/2022/12/048211), and enrolment is closed.Of 64 patients, 14 were enrolled in phase 1 (from May 11, 2021, to May 13, 2022) and 50 were enrolled in phase 2 (Dec 27, 2022, to Aug 31, 2023). The median age of the overall cohort was 44 years (IQR 27-57), and 49 (77%) of 64 patients were male and 15 (23%) were female. In phase 1, no dose-limiting toxicities occurred at doses of 2 × 106-17 × 106 CAR T cells per kg. A dose of at least 5 × 106 CAR T cells per kg was chosen for phase 2 based on a complete response in three of seven patients at this dose. The most common grade 3 or worse toxicities were haematological events: anaemia (35 [61%] of 57 patients), thrombocytopenia (37 [65%] patients), neutropenia (55 [96%] patients, and febrile neutropenia (27 [47%]) patients). There were two treatment-related deaths, one due to febrile neutropenia, immune-effector cell associated haemophagocytic lymphohistiocytosis, and septic shock, and the second due to pulmonary bleed, multiorgan dysfunction syndrome, and cytokine release syndrome. In 51 efficacy-evaluable patients (36 with B-cell lymphoma and 15 with B-cell acute lymphoblastic leukaemia), the overall response rate was 73% (37 of 51; 95% CI 59-83).Talicabtagene autoleucel had a manageable safety profile and induced durable responses in patients with relapsed or refractory B-cell malignancies. This therapy addresses an important unmet need for patients with relapsed or refractory B-cell malignancies in India.Immunoadoptive Cell Therapy (ImmunoACT) and Indian Council of Medical Research (ICMR).Copyright © 2025 Elsevier Ltd. All rights reserved, including those for text and data mining, AI training, and similar technologies.

Abstract: Clinical trials have demonstrated the efficacy of bispecific antibodies in eliciting potent antitumor responses by redirecting T cells to target cancer cells, particularly for the treatment of hematologic malignancies. However, their efficacy against solid tumors is limited by intratumoral T-cell dysfunction and inadequate persistence. The co-stimulatory domains of 4-1BB, OX40, and CD28 are most widely used in engineering chimeric antigen receptor T-cells to augment T-cell responses.In this study, we designed three co-stimulatory trispecific T cell-engaging antibodies (TriTCEs) that target Prostate-specific membrane antigen, CD3, and an additional co-stimulatory receptor(OX40, 4-1BB, or CD28). We conducted comparative profiling of the attributes of distinct co-stimulatory signals to T-cell functions in prostate cancer models.Co-stimulatory trispecific T-cell engagers enhance T-cell activation, proliferation, and display tumor cell-killing activity in vitro. These trispecific antibodies further boosted antitumor activity in humanized mouse xenograft models and increased the infiltration of CD45+ immune cells into solid tumors. Specifically, TriTCE-4-1BB and TriTCE-CD28 selectively promoted the expansion of effector memory T cells and increased the presence of CD4+ T cells more than TriTCE-OX40. T cells stimulated with TriTCE-4-1BB exhibited reduced exhaustion. Furthermore, T cells treated with co-stimulatory trispecific antibodies demonstrated enhanced metabolic activity characterized by increased oxidative phosphorylation and elevated glycolysis.Collectively, incorporating co-stimulatory receptor targeting domains represents a potentially effective strategy to unlock the full therapeutic potential of T-cell-engaging antibodies for the treatment of solid tumors.© Author(s) (or their employer(s)) 2025. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ Group.