Adoptive T cell therapy (ACT) with expanded tumor-infiltrating lymphocytes (TIL) can induce durable responses in cancer patients from multiple histologies, with response rates of up to 50%. Antibodies blocking the engagement of the inhibitory receptor programmed cell death protein 1 (PD-1) have been successful across a variety of cancer diagnoses. We hypothesized that these approaches could be combined by using CRISPR-Cas9 gene editing to knock out PD-1 in TILs from metastatic melanoma and head-and-neck, thyroid, and colorectal cancer. Non-viral, non-plasmid-based PD-1 knockout was carried out immediately prior to the traditional 14-day TIL-based ACT rapid-expansion protocol. A median 87.53% reduction in cell surface PD-1 expression was observed post-expansion and confirmed at the genomic level. No off-target editing was detected, and PD-1 knockout had no effect on final fold expansion. Edited cells exhibited few phenotypic differences and matched control functionality. Pre-clinical-scale results were confirmed at a clinical scale by generating a PD-1-deficient TIL product using the good manufacturing practice facilities, equipment, procedures, and starting material used for standard patient treatment. Our results demonstrate that simple, non-viral, non-plasmid-based CRISPR-Cas9 methods can be feasibly adopted into a TIL-based ACT protocol to produce treatment products deficient in molecules such as PD-1, without any evident negative effects.

Background Adoptive cell therapy (ACT) with tumor-infiltrating lymphocytes (TILs) has shown remarkable results in malignant melanoma (MM), while studies on the potential in other cancer diagnoses are sparse. Further, the prospect of using checkpoint inhibitors (CPIs) to support TIL production and therapy remains to be explored. Study design TIL-based ACT with CPIs was evaluated in a clinical phase I/II trial. Ipilimumab (3 mg/kg) was administered prior to tumor resection and nivolumab (3 mg/kg, every 2 weeks ×4) in relation to TIL infusion. Preconditioning chemotherapy was given before TIL infusion and followed by low-dose (2 10e6 international units (UI) ×1 subcutaneous for 14 days) interleukin-2 stimulation. Results Twenty-five patients covering 10 different cancer diagnoses were treated with in vitro expanded TILs. Expansion of TILs was successful in 97% of recruited patients. Five patients had sizeable tumor regressions of 30%–63%, including two confirmed partial responses in patients with head-and-neck cancer and cholangiocarcinoma. Safety and feasibility were comparable to MM trials of ACT with the addition of expected CPI toxicity. In an exploratory analysis, tumor mutational burden and expression of the alpha-integrin CD103 (p=0.025) were associated with increased disease control. In vitro tumor reactivity was seen in both patients with an objective response and was associated with regressions in tumor size (p=0.028). Conclusion High success rates of TIL expansion were demonstrated across multiple solid cancers. TIL ACTs were found feasible, independent of previous therapy. Tumor regressions after ACT combined with CPIs were demonstrated in several cancer types supported by in vitro antitumor reactivity of the TILs. Trial registration numbers NCT03296137, and EudraCT No. 2017-002323-25.

Almost half of the patients with metastatic melanoma obtain only short-term or no benefit at all from checkpoint inhibitor (CPI) immunotherapy. In this study, we investigated whether the immune system of patients progressing following CPI treatment was able to generate functional tumor-specific immune responses. Tumor-infiltrating lymphocytes (TILs) were isolated and expanded from metastatic melanoma lesions which progressed during or after anti-programmed cell death protein 1 (PD)-1 and anti-Cytotoxic T-Lymphocyte Antigen 4 (CTLA-4) treatment. Tumor-specific immune responses were assessed with co-culture assays of TILs and autologous tumor cells. TILs from 23 metastases of individual patients could be assessed for T cells recognition of autologous tumor cells. All metastases were progressive on or following anti-PD-1 (23/23, 100%), and the majority also after anti-CTLA-4 (17/23, 74%). Functional antitumor immune responses were detected in 19/23 patients (83%). Both CD8⁺ (in 18/23 patients, 78%) and CD4⁺ (in 16/23 patients, 70%) TILs were able to recognize autologous tumors. A large fraction of CD8⁺ TILs (median 23%, range 1.0%–84%) recognized tumor cells. This is similar to the cohorts of unselected patient populations with metastatic melanoma presented in previous studies. The localization of intratumoral immune infiltrates was heterogeneous among samples. In a phase I/II clinical trial, TILs were administered with lymphodepleting chemotherapy, pegIFNα2b and interleukin-2 to 12 patients with CPI-resistant melanoma. Out of 12 patients who previously failed CPI therapy, treatment with TILs resulted in two partial responses, of which one is ongoing. Tumor-reactive T cells appear to heavily infiltrate the tumor microenvironment of patients who failed previous CPI treatment. These patients can still respond to an infusion of unselected autologous TILs. Our results warrant further testing of novel immune re-activation strategies in melanoma patients who failed multiple CPI therapy.

Interleukin-6 blockade and radiation combined with immunotherapy may modulate the tumour microenvironment to overcome immune resistance. We assessed the efficacy of ipilimumab, nivolumab, and tocilizumab combined with stereotactic body radiotherapy (SBRT) in patients with refractory pancreatic cancer (PC). Patients with PC who had progressive disease (PD) or intolerance to gemcitabine- or fluorouracil-containing regimens were enrolled in Part A of the two-part, single-centre, phase 2 study (NCT04258150). SBRT with 15 Gy was administered on day one of the first cycle. Ipilimumab was administered (1 mg/kg every 6 weeks) for a maximum of two infusions. Nivolumab (6 mg/kg) and tocilizumab (8 mg/kg) were given every four weeks until the PD or unacceptable toxicity, or for up to one year. The primary end-point was the objective response rate, with a threshold of 15%. Twenty-six patients were enrolled and treated between April 17, 2020, and January 25, 2021. The median follow-up time at the time of data cutoff (February 7, 2022) was 4.9 months (interquartile range 2.1-7.7). No responses were observed. Five patients (19%; 95% confidence intervals [CI], 7-39) achieved a stable disease. The median progression-free survival was 1.6 months (95% CI 1.4-1.7), and the median overall survival was 5.3 months (95% CI 2.3-8.0). Overall, 19 (73%) experienced adverse events related to the treatment including two (8%) with grade 3 or higher events. The combination of ipilimumab, nivolumab, tocilizumab, and SBRT in patients with PC did not meet the prespecified criteria for expansion for full accrual.