Tumor-infiltrating lymphocyte treatment for anti-PD-1-resistant metastatic lung cancer: a phase 1 trial

Abstract

Adoptive cell therapy using tumor-infiltrating lymphocytes (TILs) has shown activity in melanoma, but has not been previously evaluated in metastatic non-small cell lung cancer. We conducted a single-arm open-label phase 1 trial ( NCT03215810 ) of TILs administered with nivolumab in 20 patients with advanced non-small cell lung cancer following initial progression on nivolumab monotherapy. The primary end point was safety and secondary end points included objective response rate, duration of response and T cell persistence. Autologous TILs were expanded ex vivo from minced tumors cultured with interleukin-2. Patients received cyclophosphamide and fludarabine lymphodepletion, TIL infusion and interleukin-2, followed by maintenance nivolumab. The end point of safety was met according to the prespecified criteria of ≤17% rate of severe toxicity (95% confidence interval, 3-29%). Of 13 evaluable patients, 3 had confirmed responses and 11 had reduction in tumor burden, with a median best change of 35%. Two patients achieved complete responses that were ongoing 1.5 years later. In exploratory analyses, we found T cells recognizing multiple types of cancer mutations were detected after TIL treatment and were enriched in responding patients. Neoantigen-reactive T cell clonotypes increased and persisted in peripheral blood after treatment. Cell therapy with autologous TILs is generally safe and clinically active and may constitute a new treatment strategy in metastatic lung cancer.

Extended Data Fig. 1

Clinical follow-up after initial nivolumab. a, Sum of tumor diameters per RECIST v1.1 over time measured by serial CT scan. Patients with evidence of tumor enlargement or new lesions within 10 weeks of initial nivolumab were treated with TIL infusion. All enrolled patients (n = 20) are included. b, Clinical follow-up of patients after initial nivolumab with clinicopathologic features in a Swimmer’s plot. Pt 04 received bridging crizotinib due to rapid progression on nivolumab. Abbreviations: TPS denotes tumor proportion score using 22C3 PD-L1 antibody. TMB; estimated tumor mutation burden based upon exome sequencing. mut/MB mutations per mega-base. ‘Ex’ denotes exon, and ‘Δ’ denotes mutation conferring exon deletion. Smoking packyears represents self-reported cigarette packs per day multiplied by total years.

Extended Data Fig. 2

Autologous reactivity screening of TIL cultured from individual tumor fragments in combination with baseline tumor cell suspensions. Shown are the interferon-γ enzyme-linked immunosorbent assays conducted without (a) and with (b) CD3 bead positive controls. Each graph is labeled with the trial patient ID. The x-axis represents the TIL fragment selected for testing. Each fragment number represents an autologous T cell line generated from a unique tumor morsel cultured in IL-2 and media between 3 to 5 weeks. Each culture is labelled by its original fragment identification number, from 48 total per patient. Post-decimal numerals indicate the tumor number, if multiple tumor sites were harvested for the same patient. An asterisk denotes whether the fragment was deemed autologously reactive. Shown is the mean ± SEM of triplicate wells from a single ELISA experiment. Each bar denotes one biologically independent culture sample (n = 1). “≤10” denotes a sample with absorbance reading below the lowest point of the standard curve performed for each assay. “TIL Ctrl” denotes a TIL culture derived from an unrelated patient to serve as a positive control for the CD3 beads and a negative control for autologous substrate. Allogeneic cancer cell lines were selected as an optional control based on partial HLA class I allele compatibility with the patient. Patient ID 12 did not have sufficient TIL growth for manufacture, and ID 26 did not have sufficient tumor for autologous reactivity testing.

Extended Data Fig. 3

Feasibility of TIL harvest and intermediate-dose interleukin-2 infusion for all patients. a, Total hours of length of stay (LOS) for inpatient admissions after TIL harvest surgery. Two patients had postoperative airleak requiring multiple days of inpatient observation. All enrolled patients (n = 20) are included. b, The total duration of infusional IL-2 for all 16 patients after TIL treatment. Patients received TIL infusion on Day 0, followed by continuous IL-2 infusion beginning 12 hours later at a dose of 18 million international units (MIU) per m2 over 6, then 12, and then 24 hours followed by 4.5 MIU/m2 over 24 hours for 3 consecutive days. c, Total inpatient length of stay for all patients. Some patients were discharged to outpatient between cyclophosphamide admission and TIL admission.

Extended Data Fig. 4

Treatment-emergent adverse events reported with cyclophosphamide, fludarabine, TIL, and IL-2. a, Adverse events grouped by National Cancer Institute preferred term occurring in 20% or more of patients attributable to cyclophosphamide, fludarabine, TIL, or IL-2. All TIL treated patients (n = 16) are included. b, All adverse events grouped by date of onset within 3 months of TIL. Patients with multiple events for a given term are counted once, using the maximum grade under each preferred term. Abbreviations: AE denotes adverse events; AST denotes aspartate aminotransferase; ALT denotes alanine aminotransferase; IL-2, interleukin-2.

Extended Data Fig. 5

Change in lymphocytes and neutrophils with lymphodepletion, TIL, and IL2. a, Initial peak followed by recovery of peripheral blood absolute lymphocyte count. b, peripheral blood absolute neutrophil count recovered by median of 7.5 days (range 4.7 – 20.6). Shown is absolute cell count (1000 cells per mm3). TIL infusion is “Day 0”. All TIL treated patients are shown (n = 16)

Extended Data Fig. 6

Radiographic enlargement of a target lesion in patient 14. Patient had biopsy-proven lung adenocarcinoma biopsy proven from a lymph node metastasis and pleural mass. She had increase in her only target lesion pleural metastasis post-TIL and biopsy revealed fibrosis. Whole exome sequencing conducted on the original tumor showed over four hundred somatic mutations. Only 3 mutations could be detected in the post-TIL biopsy despite 200x depth of sequencing. She had new metastases appear 1.4 years later. Shown is a representative hematoxylin and eosin (H&E) image from 15 high-powered field views, selected by diagnostic clinical pathologists. Bar denotes approximately 100 μm. Pre-TIL biopsy comprises 5 separate preserved blocks from 1 tumor from a single procedure performed on one day. Post-TIL biopsy comprises 3 core needle samples obtained from 4 total passes from a single procedure performed on one day. Each somatic mutation count represents 1 biologically independent sample.

Extended Data Fig. 7

Overall survival from enrollment. a, Overall survival of all enrolled patients (n = 20) from date of enrollment. b, Overall survival of patients who had not received any anti-cancer systemic therapy prior to enrollment (n = 12), and c, of patients who had received at least one line of previous systemic therapy (n = 8). Hashmarks denote 95% confidence intervals for survival probability. All patients were assessed for survival through the data cut-off of 10 Sept. 2020.

Extended Data Fig. 8

In vitro expansion of autologous T-cell clonotypes after stimulation with peptide antigens. Clonotypes with significant increase using autologous T and dendritic cells from a, Patient 25 and b, Patient 9. T cells were co-cultured with autologous dendritic cells for 10 days and compared to controls wells including T cells only and no peptides. TCR Vβ CDR3 AA denotes T-cell receptor Vβ complementaritydetermining region 3 amino acid. Fold expansion is relative to baseline control without peptide.

Extended Data Fig. 9

Expression of immune checkpoint ligands and cell composition of final infused TIL product a, Expression of immune checkpoint and cell activation markers assessed by flow cytometry as a proportion of either CD4⁺ or CD8⁺ T cells (n = 18 patients with 1 biologically independent experiment per patient, bar denotes median). b, Proportion of regulatory T cells (CD4⁺ CD25⁺CD127^dim). (n = 18 patients with 1 biologically independent experiment per patient). c, TIL cell population stratified by tdistributed stochastic neighbor embedding (t-SNE) mapping of memory differentiation subsets. Defined cell populations were assigned specific colors. Each patient is labeled by Pt ID. There are 18 patients presented in total, with 1 biologically independent experiment per patient.

Extended Data Fig. 10

Phenotype and clonotype features of final infused TIL Phenotype and clonotype features of final infused TIL a, Best overall response (BORR) and proportion of tumor-specific fragments in the final product. All patients with sufficient TIL for autologous reactivity testing performance are shown (n = 18). Pt 26 had insufficient tumor digest for autologous reactivity testing, and Pt 12 had insufficient pre-REP TIL for testing. A proportion were tested for reactivity with autologous tumor suspension using ELISA for interferon-γ. Pink is the proportion of autologous-reactively cultures pooled for the final rapid expansion. Gray are either non-specific or negative cultures added to the pool, to ensure sufficient starting cell numbers. b, Patient BORR and dose of CD8⁺ or CD4⁺ cells in final expanded TIL product. All patients with final manufactured TIL are shown (n =18). Pt 30 pre-REP TIL is still cryopreserved, and Pt 12 had insufficient pre-REP TIL to manufacture. 48 minced tumor fragments were cultured in IL-2. c, Change in T cell clonotypic composition between original intratumor T cells and final infused TIL. Across patients, there were variable numbers of enriched clones in the TIL culture, with a range 13 to 1011 clones. A large proportion of clones in each patient were not detected in the baseline tumor. d, Comparison of the most prevalent one hundred clones in the manufactured TIL culture with baseline tumor, with total number shown on the x-axis and overlap in middle. High repertoire turnover during TIL culture was observed in most samples. Several cultures shared no high frequency clones with their baseline tumors. e, Productive clonality Simpson clonality increased from baseline tumor to final cultured TIL (Exact p = 0.000013, two-sided Wilcoxon signed rank test). Shown is the median and 95% confidence interval overlaying the individual values. All patients with manufactured TIL are shown (n =19). Abbreviations: NT; not treated. NE; not evaluable. uPR, unconfirmed partial response.

Figure 1.. Schematic representing study design and patient disposition.

a, Clinical trial schema. Day count is relative to TIL infusion. b, Patient disposition per Consolidated Standards of Reporting Trials (CONSORT) guidelines. Of the 16 patients treated with TIL, 2 of them had initial response to nivolumab, followed by progressive disease prior to receiving TIL therapy. Abbreviations: TKI denotes tyrosine kinase inhibitor; IL-2, interleukin-2; TIL, tumor-infiltrating lymphocyte treatment; PD, progressive disease; q w, every week; ID, patient number.

Figure 2:. Clinical activity of TIL and patient survival.

a, Features of patients who were treated with TIL and their clinical outcomes (n = 16). b, Change in sum of tumor diameters, relative to Week −1 prior to TIL. Non-evaluable patients (ID 03, 13, 15) are not shown. Pt 14, designated as radiographic “PD” post-TIL, had biopsy of only target lesion showing fibrosis with no tumor cells at the time of progression. c, Waterfall plot showing best overall change in sum of diameter of tumor lesions. Change in sum of tumor diameters by RECIST is compared to Week −1 prior to TIL. Pt 14, designated as radiographic “PD” post-TIL, had biopsy showing of only target lesion showing fibrosis with no tumor cells. All patients evaluable with a post-TIL CT scan are included (n =13). Non-evaluable patients (ID 03, 13, 15) are not shown. “*” denotes initial PR with nivolumab, followed by biopsy-proven unequivocal PD (ID 07, 09), ‡ Unconfirmed PR, subsequent non-target PD (ID 02, 05, 31); §§ Had 10 mm increase in target lesion on prior nivolumab. (ID 08).d, Overall survival of all treated patients from date of TIL infusion (n = 16 patients). Hashmarks denote 95% confidence intervals. Median follow-up of 1.6 years (range 0.5 – 2.9) by reverse Kaplan-Meier method. Abbreviations: TPS denotes tumor proportion score by 22C3 antibody immunohistochemistry; IL-2, interleukin-2 and TIL, tumor-infiltrating lymphocyte treatment. TMB, estimated tumor mutation burden; mut/MB, mutations per megabase. ‘Ex’ denotes exon, and Δ denotes exon deletion. Smoking pack-years represents self-reported cigarette packs per day multiplied by total years.

Figure 3.. Examples of complete responses mediated by TIL recognizing tumor antigens.

a, Patient (ID 25) with an EGFR ^ΔEx19 tumor had progressive metastases with nivolumab, followed by complete response to TIL. The sum of radiographic target lesions is shown over time with representative contrast-enhanced axial CT images. b, IFN-γ spot formation after co-culture of her post-TIL T cells with her autologous dendritic cells pulsed with synthesized long peptides displaying tumor antigens. Five proteins are shown, of 104 tested, mean ± SEM of 3 plates over 2 experiments. c, Absolute number of antigen-specific T cell receptor clonotypes within the peripheral blood after TIL. The absolute number and proportion increased after TIL, and then gradually decayed. Data is derived from a total of 10 serial blood samples. The proportion of antigen-specific clones in her infused TIL product is shown in the pie chart. d, Patient (ID 09) is a former smoker with lung adenocarcinoma. She had partial response to nivolumab followed by enlargement of metastatic lymph nodes and a new biopsy-proven rib soft tissue metastasis 10 months later. She was then treated with TIL and had a complete response with ongoing absence of radiographic target lesions. Representative coronal contrast-enhanced CT images are shown over time. e, Co-culture of either CD4⁺ or CD8⁺ T cells sorted from the final infused TIL with autologous dendritic cells and custom peptides corresponding to tumor neoantigens elicited reactivity. Three positive peptides are shown, of 85 tested. Mean ± SEM of 3 plates over two experiments. f, Absolute number of antigen-specific clonotypes in the peripheral blood increased and then gradually decayed after the TIL infusion. Data is derived from a total of 13 serial blood samples. Abbreviations: TRVβ, T cell receptor variable beta chain; MAGE, melanoma associated gene; IFN, interferon; Pt, patient.

Figure 4.. Summary of tumor-specific antigens tested for all patients.

Top panel shows the total sum of unique genomic alterations tested for each patient. Bottom panel shows the tumor-specific antigens which were detected by autologous T cells. Positive antigens were assessed for T cell reactivity by synthesis of the corresponding recombinant peptides, and identification of IFN-γ colony formation by ELISpot, relative to controls with autologous T cells and dendritic cells only. Mean spot forming units per positive Ag are shown, with n = 2 – 5 tests per antigen. Cultured TIL was used to assess antigen reactivity for all patients except 02, 25, 32, 33 due to high IFN-γ background of the cultured TIL. In these instances, autologous T cells isolated from post-TIL peripheral blood were used. Abbreviations: IFN, interferon; Pt, patient; TIL, tumor-infiltrating lymphocyte; Ag, antigen.

Figure 5.. Change in phenotype and genotype of peripheral T cells after TIL infusion.

a, Increase in circulating subsets of polyfunctional CD8⁺ or CD4⁺ T cells at post-infusion timepoints. *Friedman test using mixed-effects model (REML) with stacked matching with Geisser-Greenhouse correction. Shown is the multiplicity adjusted, two-sided p value using Dunnett’s control for multiple comparisons, with family-wise alpha of 0.05. Exact p value 0.0000008. Stacked bars denote mean ± SEM of available dataset with 12 tested patients. b, Increase in peripheral CD45RA⁻CCR7⁻ effector memory T cells at post-infusion timepoints for all patients with available timepoints (n = 15) with 8 serial timepoints collected per patient. Stacked area curves denote mean ± SEM percentage of CD8⁺ or CD4⁺ T cells. Lineage definitions: Naïve T cells, CD45RA⁺CCR7⁺CD95⁻; central memory, CCR7⁺CD45RA⁻; effector memory, CCR7^-CD45RA⁻; stem cell-like memory, CCR7⁺CD45RA⁺CD95⁺; effector T cells, CCR7^-CD45RA⁺”. d, Overlap of TRVβ chain productive rearrangements between final infused TIL product and peripheral blood T cells at indicated time-points, using Morasita’s index. Bars denote mean ± SEM of all patients with available timepoints for analysis (n = 16). Abbreviations: TRVβ, T cell receptor variable beta chain; SEM, standard error; TIL, tumor-infiltrating lymphocyte infusion; nivo, nivolumab; MIP, macrophage inflammatory protein; IFN, interferon. REML, restricted (or residual, or reduced) maximum likelihood; CCR, chemokine receptor.