Tumor‐Infiltrating Lymphocyte Adoptive Cell Therapy With PD‐1 Blockade in Pediatric Chemotherapy‐Resistant Osteosarcoma

To the editor; We describe the case of a 13-year-old female patient with chemotherapy-resistant metastatic osteosarcoma. With no remaining treatment options, an experimental approach with tumor-infiltrating lymphocytes (TILs), followed by high-dose (HD) IL-2 and anti-PD-1 therapy, was pursued. TILs were successfully expanded, and treatment was feasible and well tolerated, but no clinical response was observed. This case illustrates the feasibility and limitations of TIL therapy in pediatric osteosarcoma and the need for immune profiling to identify likely responders. Osteosarcoma is the predominant malignant mesenchymal tumor of bone in children and adolescents [1]. Although overall survival for localized disease is approximately 70%, prognosis for metastatic or relapsed osteosarcoma remains poor, with overall survival near 20% [2]. Standard treatment based on the EURAMOS-1 trial includes neoadjuvant and adjuvant MAP chemotherapy—HD methotrexate, doxorubicin, and cisplatin—combined with complete resection of the primary tumor and resectable metastases. For unresectable or relapsed disease, no established standard treatment exists [3, 4]. Adoptive cell therapy (ACT) with TILs utilizes patient-derived polyclonal tumor-infiltrating T cells, expanded ex vivo and infused after lymphodepleting chemotherapy, followed by in vivo stimulation with IL-2. TIL-ACT has demonstrated superior efficacy over ipilimumab in advanced melanoma in a randomized phase III trial leading to its FDA approval in 2024 [5]. Feasibility has also been demonstrated in other solid tumors, including soft tissue sarcomas [6, 7]. Combining TIL therapy with PD-1 blockade is rationalized by the limited efficacy of checkpoint inhibitors alone in sarcomas. The SARC028 evaluating pembrolizumab monotherapy in sarcoma showed minimal responses, likely due to T cell exhaustion and a limited pool of tumor-reactive T cells [8, 9]. Thus, our approach aimed to supply activated tumor-reactive T cells while overcoming PD-1-mediated immune suppression. A retrospective study of ACT-TIL in combination with nivolumab in patients with osteosarcoma reported encouraging responses (objective response rate 36.7%, disease control rate 80%) but omitted lymphodepletion and IL-2 support [10], both associated with improved efficacy through elimination of regulatory T cells and cytokine sinks as well as promotion of TIL proliferation, respectively [11, 12]. To maximize therapeutic potential, we therefore used a melanoma TIL regimen, including lymphodepletion and HD IL-2 [5]. In March 2023, our patient was diagnosed with metastatic osteosarcoma and was treated as outlined in Figure 1. Adjuvant chemotherapy was completed in December 2023; however, multiple new lung metastases were detected in January 2024. Following salvage therapy with ifosfamide, carboplatin, etoposide, and regorafenib—under compassionate use—temporary authorization for TIL-ACT followed by PD-1 blockade with nivolumab was granted by the Swiss authorities. For TIL manufacturing, 15 metastatic lung lesions (≤5 mm3) were procured. Tumor fragments (1–5 mm3) were cultured for 10 days with IL-2 (6000 IU/mL), followed by cryopreservation. After thawing, TILs were expanded with IL-2 (3000 IU/mL), anti-CD3 antibody (30 ng/mL), and irradiated PBMCs (1:200 TIL:feeder) for 7 days in static culture vessels and 7 days in a rocking bioreactor. The final product contained 52 × 109 cells (88.2% viability, 98% CD3+) (Table S1). During TIL manufacturing, lymphodepletion was initiated with cyclophosphamide (60 mg/kg/day, days −7 to −6) and fludarabine (25 mg/m2/day, max 50 mg, days −7 to −3). Fresh TILs were infused on day 0, followed by HD IL-2 (600,000 IU/kg every 8 h). IL-2 was discontinued after six doses due to cytokine release syndrome, which was managed with vasopressors and one dose of tocilizumab. Nivolumab (3 mg/kg) consolidation was started 3 weeks later. Adverse events (AEs), graded according to NCI common terminology criteria for AEs (CTCAE 5.0), are summarized in Table 1. No unexpected or unmanageable toxicities occurred. Hematological toxicities included anemia, lymphopenia, and neutropenia from lymphodepletion. No nivolumab-related grade ≥2 AEs occurred. 3 months post-TIL transfer, imaging demonstrated disease progression. HD IL-2 treatment Lymphodepletion The poor response observed in this case may be attributable to characteristics of osteosarcoma such as variable PD-L1 expression, low MHC class I expression, sparse lymphocyte infiltration, dense stroma, reduced adhesion molecules (ICAM-1, LFA-3), and an immunosuppressive tumor microenvironment characterized by factors such as TGF-β [13-15]. Furthermore, prior chemotherapy may have reduced the available T cell pool for TIL manufacturing. In the future, immunogenomic profiling of tumor characteristics may help identify patients who are more likely to benefit from such therapeutic approaches. Immune classification of sarcomas based on B-cell-rich tertiary lymphoid structures has also been proposed as a predictive biomarker for response [16]. Importantly, TIL therapy may also be effective in more immunogenic pediatric solid tumors such as alveolar soft part sarcoma [17] or undifferentiated pleomorphic sarcoma [18, 19]. Beyond TIL therapy, osteosarcoma expresses antigens, including MHC-dependent targets MAGE, SART1, SART3, and PBF [20], and MHC-independent targets, such as B7-H3 (CD276) [21] and disialoganglioside GD2 [22, 23], enabling T cell receptor T cell (TCR-T) and chimeric antigen receptor T cell (CAR-T) approaches. Hybrid strategies such as CAR-transduced TILs may combine natural tumor tropism with enhanced antigen specificity [14]. In conclusion, this case demonstrates the feasibility and safety of ACT with TILs, including lymphodepletion and HD IL-2 in combination with PD-1 blockade, in a pediatric patient with metastatic osteosarcoma. The absence of clinical response highlights the challenges of immunotherapy in this disease and underscores the need for innovative, personalized approaches to improve outcomes. We thank the patient for allowing us to report the clinical information and data. We also thank Anke Wixmerten and the team of the GMP Facility for Advanced Therapies for their support, and Jessica Häusel, Melanie Koch, and Matthias Reist for their involvement in GMP manufacturing. The patient was treated under a temporary authorization by Swissmedic and local ethical committee (EKNZ). The patient and her parents signed an informed consent allowing the use of the data for publication. All procedures were conducted in accordance with with the Declaration of Helsinki and its later amendments. The authors declare no conflicts of interest. Table S1: Batch Data Summary Supplementary Figure: Batch Data Summary Tumor-infiltrating lymphocyte (TIL) manufacturing batch data are summarized, including all parameters meeting the predefined acceptance criteria required for product release. Fold expansion and population doubling rate achieved during the rapid expansion phase are also highlighted. 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