All patients provided informed consent in accordance with the Declaration of Helsinki. The ETC trial (NCT04177021) was approved by the Fred Hutchinson Cancer Research Center (FHCRC) Institutional Review Board (IRB). The primary objective was to assess the feasibility, safety and toxicity of treating of NY-ESO-1–specific ETC. Tolerability based on CTCAE V.4.0 was the primary endpoint. Response based on RECIST V.1.1 at 8–10 weeks and T-cell persistence based on tetramer (tet+) staining were secondary endpoints. Eligibility included advanced or metastatic SS or MRCL with at least one prior line of therapy, expression of HLA A*0201, ECOG performance status ≤1 and NY-ESO-1 expression in >25% of tumor cells by immunohistochemistry (for complete eligibility criteria, see online supplemental figure 1). Patients were labeled Cy1–Cy4 based on order of enrollment. Details regarding cell generation have been previously reported.9 Baseline imaging was performed approximately 2 weeks prior to infusion and an additional scan was performed 4 weeks afterwards. After their 8–10 week scan, patients were followed per standard of care with additional research blood collections. The patient who received ACT at the NCI (labeled “NIH”) consented for a blood/tissue collection protocol registered with the FHCRC IRB. The patient treated at UCLA (labeled “UCLA”) provided informed consent at UCLA and de-identified PBMC was analyzed at FHCRC under an IRB-approved minimal risk study.

Multicolor staining including Major Histocompatibility Complex (MHC) tetramers was as previously described.9 For cytokine staining, cells were stimulated with NY-ESO-1_157–165 peptide (10 µg/mL) for 1 hour followed by addition of 1× Brefeldin A (BioLegend). For intracellular markers, cells were fixed using a 1:3 ratio of fixation and permeabilization (eBioscience). Data were collected using the BD FACS Symphony and analyzed with FlowJo. See online supplemental table 1 for a full list of antibody and clones.

RNA from sorted CD8+tetramer+ (tet+) was extracted using Qiagen AllPrep DNA/RNA Micro Kit. Gene-expression profiling was performed on the Affymetrix Clariom D Pico assay platform. Microarray data were normalized and analyzed by Limma. Repeated measure was applied to all patients to account for only time-dependent change. Gene sets were obtained from MSigDB Collections.

TCR Vβ sequencing and normalization was performed on tet+ cells sorted from patients’ PBMCs by Adaptive Biotechnologies (Seattle, WA, USA). The number of total cells, T cells, the T-cell fraction, the number of unique rearrangements and clonality were calculated for each sample as previously described.13

Serum samples were collected before infusion and at different times post-infusion, then stored at −20°C. Samples were analyzed in duplicates at the same time using a Luminex microbead assay.

Tumor specimens were cut into 1–2 mm fragments and cultured with 2000 U/mL IL-2 and cryopreserved. To liberate additional T cells from tumor, some frozen fragments were thawed, incubated in an enzymatic cocktail containing Type IV collagenase, DNase and hyaluronidase for 30 min at 37°C, then pressed through a 70 mm mesh cell strainer.

PBMCs (1×10⁵) from each patient were cultured in the presence of NY-ESO-1_157–165 peptide (10 µg/mL) and in STEMCELL-XF T expansion medium conditioned with IL-2, IL-15 and in some cases anti-PD-1. Half-media changes with conditioned media were performed on every alternate day until day 8 when PBMCs were harvested for flow cytometry. For the 3D culture assay, GFP-transduced 1765 MRCL cells were embedded in Collagen Type I in organoid chips (AIM Biotech) for coculture assays.14 Chips were imaged using the Leica SP8 microscope and analyzed using Imaris software (see online supplemental table 2 for information about reagents, concentrations and details regarding the 3D culture assay).

For multiple groups, statistical significance was calculated using one-way ANOVA, followed by Dunnet’s multiple comparison test to compare two groups within multiple groups. For only two groups, Student’s t-test was applied. ****p≤0.0001; ***p≤0.001; **p≤0.01; *p≤0.05.

Patient demographics are included in table 1. Four patients were treated with NY-ESO-1–specific ETC at target cell doses of 10¹⁰/m² on day 0 after receiving Cy (2 g/m²/day ×2 days) on days −3 and −2 as lymphodepleting conditioning (figure 1A). No unexpected, attributable toxicity was seen and all significant toxicity (grade ≥3) was attributed to Cy (complete toxicity data are included in online supplemental table 3). Patient Cy1 had small volume lung disease with only one RECIST-evaluable lesion with stable disease (SD), and slight regression from 11.5 mm to 10.9 mm following ACT. This lesion was resected 3 months after infusion allowing the patient to be free of detectable disease, but recurred after 1 year. Cy2 started with rapidly progressive disease (PD), but had a 20% reduction in a liver lesion at his 4-week scan (figure 1B) with SD overall. Cy3 had rapidly progressing pulmonary metastases pre-treatment, with baseline imaging demonstrating 62% increase versus prior scan 2 months earlier. The 4-week post-ACT scan showed SD with some smaller tumors. A subsequent scan showed PD and the patient went on to receive chemotherapy and eventually metastatectomy. Cy4 had rapidly progressing bulky disease but had SD with some tumors regressing post-ACT and improvement in his pain allowing him to discontinue opioids (clinical summaries are provided in online supplemental figure 2).

Although each patient other than Cy1 had confirmed PD by their 8–10 week CT scan, all had evidence of anti-tumor activity in at least one tumor site at the 4-week scan, suggesting temporary activity of the infused cells given that these patients had prior treatment with ifosfamide and thus were unlikely to respond to Cy. The cohort was stopped after four patients, as alterations to the conditioning regimen were planned (NCT02319824). Retention of viable tumor with homogeneous NY-ESO-1 expression was confirmed for both patients with tumors resected post-ACT (Cy1 and Cy3; Cy3 shown in figure 1C). Serum IL-2 and IL-15 dropped after an initial post-Cy spike (IL-15 shown in figure 1D, IL-2 in online supplemental figure 3).

For each patient, the frequency and number of CD8+NY-ESO-1 tet+ cells in PBMC peaked at week 2 and then decreased (figure 1E and F) to low yet still detectable levels. In Cy1, whose tumor was resected 3 months after ACT, 2.9% of TIL were CD8+/tet+, 5× higher than in the PBMC at that time suggesting trafficking to the tumor (figure 1G). The early peak of dominant transferred clones was confirmed in TCRβ sequencing of PBMC at 1 day, ~2 weeks, ~1 month and ~2 months post-ACT (figure 1H). TCRβ sequencing was performed on tet+ cells sorted from similar time points. In Cy2 and Cy4 (Cy2 shown in figure 1I), the dominant clone in this tet+ sorted population remained dominant for at least 8–10 weeks. In Cy1 and Cy3, the dominant clone waned, and minority clones increased in frequency in the tet+ population (online supplemental figure 4).15

Consistent with prior reports,10 16 transferred cells in all patients predominantly had a memory phenotype (figure 2A and B)17 with very few cells that were double positive for CD39 and CD103. While few cells expressed PD-1, we also tested for Tim-3, an exhaustion marker that also suggests antigen experience (online supplemental figure 5), and there was a non-statistically significant trend toward higher expression on tet+ cells compared with tet− controls (most pronounced in Cy1, figure 2C and D). Given that at least two patients retained NY-ESO-1 expression post-ACT, we expected to observe proliferation and expansion of persisting cells. However, Ki67 expression was much lower on tet+ cells compared with tet− controls (figure 2D) suggesting a lack of proliferation. We performed gene expression analysis on flow sorted tet+ cells from 1 day, ~2 weeks and ~2 months after infusion (phenotype of infusion products has been previously described).9 Gene sets related to TCR signaling, T-cell activation, cytotoxic capacity and IL-2 pathway were more highly expressed at week 2 compared with weeks 8–10 post-infusion suggesting that transferred T cells lacked TCR stimulation post-ACT (figure 2E).

Given that rare persisting cells failed to proliferate despite the presence of confirmed antigen-expressing tumor, we hypothesized that these cells might be stimulated with IL-15, which has been observed to stimulate inhibited memory T-cell populations.18 To test this, we cultured PBMCs from post-infusion samples with NY-ESO-1_157–165 peptide and low-dose IL-2 alone, or with IL-15. The addition of low-dose IL-15 led to a significant increase in expansion of tet+ cells compared with control group by day 9 (figure 3A). Although IL-15 also induced expansion of CD8+ cells, the expansion of tet+ cells was significantly higher (figure 3B).

We also confirmed this effect in PBMCs from patients with SS treated with NY-ESO-1 TCR-engineered products at UCLA and the NIH who also had a low number of persisting tet+ cells.5 12 We found that IL-15 expanded tet+ cells from tumor-infiltrating lymphocytes (TILs) isolated from post-treatment biopsy from Cy1 (figure 3A). Neither anti-PD-1 nor higher doses of IL-15 further enhanced this effect (online supplemental figure 6). In patients Cy2 and Cy4, the effect of IL-15 in inducing T-cell expansion was higher in samples from intermediate time points compared with early or late time points suggesting that the effect of IL-15 may be time dependent for some patients (figure 3C).

To test if IL-15 increased the effector function of T cells, we stimulated cells with NY-ESO-1_157–165 peptide following 9-day culture with IL-2 or with IL-2 and IL-15 and performed intracellular cytokine staining for IFNγ and TNFα. The number of cells positive for both IFNγ and TNFα was significantly higher in IL-15 supplemented cultures (online supplemental figure 7), and we confirmed these results in samples from patients treated with TCR-engineered products at UCLA, and the NIH, as well as TIL from Cy1 (figure 3D).

To more closely approximate in vivo biology, we performed T-cell tumor killing assays using a 3D culture system in which tumor cells and T cells are embedded in a collagen matrix lined by endothelial cells. Tet+ cells cultured with IL-15 had greater than twofold increase in their killing efficiency as compared with the control group. By 48 hours, the number of IL-15-treated tet+ cells were significantly higher than the control group, despite starting with the same number of cells suggesting that T cells continued to expand into the chips post-transfer (figure 3E; online supplemental figure 8). These results were observed in both products from patients treated at UCLA and the NIH.