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.
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TRV-β sequencing of tumor and peripheral T cells as the project ‘TIL trial for NSCLC’ are available in the immuneACCESS free public database at https://clients.adaptivebiotech.com/immuneaccess. Tumor WES and RNA-seq raw data files are available on the public National Institutes of Health (NIH) dbGaP data browser (https://www.ncbi.nlm.nih.gov/projects/gapprev/gap/cgi-bin/preview1.cgi?GAP_phs_code=UgsrhvZ3dGC4uQSh; accession code phs002486.v1.p1). Raw data on expression, allelic frequency and predicted MHC affinity for all identified neoantigens are provided in the Excel spreadsheet in Supplementary Table 4. The study protocol is uploaded as a supplementary file. In the event that source data are not inferred from specified tables/figures, they are available upon request in a comma-delimited format from the study authors.
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This work was supported by a Catalyst Award Clinical Trial grant through the Stand Up to Cancer Foundation (SU2C-AACR-CT04-17; to E.B.H., S.J.A.); Barbara Bauer Prelude to a Cure Foundation grant to B.C.C.; ER Squibb for nivolumab supply; Iovance Biotherapeutics as a research grant, Clinigen for aldesleukin supply, an NCI Cancer Center Support Grant (P30-CA076292; to E.B.H.) and 2018 Young Investigator award from Adaptive Biotechnologies to B.C.C. Stand Up 2 Cancer is a program of the Entertainment Industry Foundation administered by the American Association for Cancer Research. We thank the patients and their families who participated in this research; members of the Moffitt Cell Therapies Facility, including L. Kelley and C. Cox for cell manufacturing; C. Yang and T. Mesa at the Moffitt Molecular Genomics Facility, M. Wloch, N. Smith and K. Fahrner at the Moffitt Tissue Core and P. Cano at the Moffitt Histocompatibility Leukocyte Antigen laboratory; T. Boyle and G. Zhang for assistance with clinical specimens; C. Ulge for grant administration; M. Avedon for translational project management; and other members of our clinical and laboratory research teams. Cell sorting for this project was performed on instruments in the Moffitt Flow Cytometry Core. Sequencing was performed by the Moffitt Molecular Genomics and Proteomics & Metabolomics Core Facilities (supported in part by NCI Cancer Center Support Grant P30-CA076292). Clinical care and trial coordination was provided by the Moffitt Immune and Cellular Therapies department. We thank B. Banbury for assistance with the graphs of T cell clonotypes.
Moffitt Cancer Center (Principal Investigator B.C.C.) was the sponsor of the trial. The trial was primarily supported by Stand Up to Cancer Foundation through the AACR Catalyst grant mechanism. Nivolumab was supplied by ER Squibb & Sons. Aldesleukin (IL-2) was supplied by Clinigen Group. Funding to the Moffitt Cell Therapies Facility was provided by Iovance Biotherapeutics. The companies played no other role in the study or report. B.C.C. has received speaking fees from AstraZeneca, ARIAD Pharmaceuticals and Hoffmann-La Roche and consultant fees from Xilio, Achilles, ER Squibb, Hoffmann-La Roche, AstraZeneca, AbbVie, KSQ Therapeutics, GlaxoSmithKline, Gilead Sciences, Celgene. B.C.C. has received research funding from NeoGenomics Laboratories and Adaptive Biotechnologies and has a patent application (WO2020263919A1). A.A.C. has received speaking fees from Genentech, Merck, Celgene and Takeda; consultant fees from Amgen, Jazz, AstraZeneca, Pfizer, Novartis, AbbVie and BMS; and research funding from AstraZeneca, Novartis and BMS, outside the submitted work. J.R.C.G. has received consultant fees from Leidos, consultant and nonfinancial support from Anixa Biosciences; consultant fees and nonfinancial support from Compass Therapeutics; and has a patent (WO2020033923A1) pending. E.B.B. has received consultant fees from Amgen and Janssen, outside the submitted work. S.K. reports nonfinancial and research financial support from BMS and AstraZeneca. A.M.L. received financial support from by Iovance Biotherapeutics. J.E.M. reports laboratory research support from Iovance Biotherapeutics for research outside the submitted work and a patent (62668246) licensed to Iovance Biotherapeutics. S.A.P.T. reports grants from Swim Across America Foundation, V Foundation, American Cancer Society Research Scholar Grant, State of Florida Bankhead-Coley Grant, Iovance Biotherapeutics, Intellia Therapeutics, Myst Therapeutics, NIH-NCI (grant nos. U54 CA193489-01A1, CA241559, U01 CA244100-01, R01 CA239219-01A1) and Provectus Biopharmaceuticals, outside the submitted work. In addition, S.A.P.T. has a patent (61/973,006 61/978,112) with royalties paid to Iovance Biotherapeutics and a patent immunotherapy targeted to tumors expressing receptors with royalties paid to Tuhura Biopharma. A.N.S. received nonfinancial support and research grants from Daiichi Sankyo, Novartis, Mersana, Genmab and Eli Lilly, outside the submitted work. A.A.S. reports grants from NCI K23, Ocala Royal Dames and Swim Across America Foundation; personal fees from Guidepoint and Gerson Lehrman Group; and consultant fees from Defined Health, Iovance Biotherapeutics, Physicians’ Educational Resource and Medscape, outside the submitted work. In addition, A.A.S. has a patent for Compositions and Methods for Improving Tumor-infiltrating Lymphocytes for Adoptive Cell Therapy (61/973,002) with royalties paid to Iovance Biotherapeutics, a patent for a Rapid Method for Culture of Tumor-infiltrating Lymphocytes from Core Needle Biopsies of Solid Tumors and a patent for Tumor-infiltrating Lymphocytes and Stapled Peptoid Peptide Hybrid Peptidomimetics (16/157,174). J.K.T. has grant support through P30-CA76292 and a patent pending for a Large Data Set Negative Information Storage Model. C.W. has a patent application filed for Generation of T Cell Receptors (TCRs) that React to Neoantigens. S.J.A. has received advisor fees from Achilles, Amgen, AstraZeneca, ER Squibb, Caris Life Sciences, Celsius Therapeutics, G1 Therapeutics, GlaxoSmithKline, Memgen, Merck & Co., Nektar Therapeutics, RAPT Therapeutics, Venn Therapeutics, Glympse, EMD Serano and Samyang Biopharm USA; and research funding from Cellular Biomedicine Group. The remaining authors declare no competing interests.
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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 pack-years 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 air-leak 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.
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).
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.
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 complementarity-determining 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+CD127dim). (n = 18 patients with 1 biologically independent experiment per patient). c, TIL cell population stratified by t-distributed 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.
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.
Supplementary Tables 1 and 2 and Appendixes 1 and 2.
Source data for neoantigen prediction priority (Table 3) and reactive peptide neoantigens with their predicted binding affinities (Table 4) as tabs of the spreadsheet.
Clinical trial protocol.
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Creelan, B.C., Wang, C., Teer, J.K. et al. Tumor-infiltrating lymphocyte treatment for anti-PD-1-resistant metastatic lung cancer: a phase 1 trial. Nat Med 27, 1410–1418 (2021). https://doi.org/10.1038/s41591-021-01462-y
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