Lifileucel tumor-infiltrating lymphocyte cell therapy in patients with unresectable or metastatic mucosal melanoma after disease progression on immune checkpoint inhibitors

Mucosal melanoma (MM) is a rare melanoma that affects the mucous membranes of the gastrointestinal, respiratory, and genitourinary tracts [1]. In contrast to cutaneous melanoma (CM), MM occurs in body areas without sun exposure and is more difficult to detect, often overlooked until nodal or metastatic involvement [2]. The molecular profile of MM is distinct, with a lower mutational burden and higher degree of chromosomal aberrations than CM, potentially affecting treatment strategies [3]. Patients with MM typically receive the same immunotherapy as patients with CM and are not candidates for BRAF/MEK inhibition, an option for many patients with CM [1]. However, standard-of-care therapies and immune checkpoint inhibitors (ICIs) are associated with poor outcomes in patients with MM [4-6].

C-144-01 (NCT02360579) was a phase II multicenter, multicohort study of lifileucel autologous tumor-infiltrating lymphocyte (TIL) cell therapy in patients with advanced (unresectable or metastatic) melanoma, including MM, whose disease progressed on or after anti-programmed cell death protein-1/programmed cell death-ligand 1 (PD-1/PD-L1) therapy [7]. With median follow-up of 27.6 months (range, 0.2+ to 48.7 months), lifileucel demonstrated an Independent Review Committee (IRC)-assessed objective response rate (ORR) of 31.4% in heavily pretreated patients with advanced melanoma [7]. Lifileucel was approved by the US Food and Drug Administration in 2024 for the treatment of adults with unresectable or metastatic melanoma previously treated with a PD-1-blocking antibody, and if BRAF V600 mutation was positive, a BRAF inhibitor with or without a MEK inhibitor.

Herein, we report outcomes with lifileucel in patients with advanced MM in the C-144-01 study. Of 153 ICI-refractory patients who received lifileucel and were analyzed for efficacy in pooled cohorts 2 and 4, 12 (7.8%) had MM (Supplementary Figure S1) [7]. The median age of the patients with MM was 61.5 years and 6 (50.0%) were female (Supplementary Table S1). The median number of prior therapies was 2. Five patients (41.7%) had elevated lactate dehydrogenase. Disease burden was greater than in the overall population, with a median sum of diameters (SOD) of target lesions of 118.9 mm and a median of 6 target and nontarget lesions. Ten patients (83.3%) were primarily refractory to anti-PD-1/PD-L1 therapy. Baseline and disease characteristics for the entire study population are provided for comparison (Supplementary Table S1).

Patients with ≥1 resectable lesion underwent tumor resection, and samples were shipped to a centralized facility for the 22-day lifileucel manufacturing process (Supplementary Material and Methods). Fifteen patients with MM had tumor resection, with median total manufactured viable TIL cells of 25.90 × 10⁹ (range, 0.04-72.00 × 10⁹). Three patients did not receive TIL infusion owing to disease progression (1 [6.7%]), TIL not available (manufacturing failure, 1 [6.7%]), or start of new anticancer therapy (1 [6.7%]). Twelve patients received nonmyeloablative lymphodepletion (cyclophosphamide 60 mg/kg for 2 days; fludarabine 25 mg/m² for 5 days), a single lifileucel infusion, and up to 6 doses of high-dose interleukin-2 (IL-2) at 600,000 IU/kg. Among the 12 patients who received TIL infusion, 6 had tumor tissue procured from lymph nodes and 2 from the lungs. Median (range) number of TILs infused was 26.1 × 10⁹ cells (3.3-72.0 × 10⁹) and number of IL-2 doses was 5.5 (3.0-6.0).

With median follow-up of 35.7 months (range, 27.9-49.6 months) (data cutoff: July 15, 2022), the IRC-assessed ORR (primary endpoint) was 50.0% in patients with MM (6/12; 95% confidence interval [CI], 21.1%-78.9%), with 1 (8.3%) complete response and 5 (41.7%) partial responses (Supplementary Table S2). Nine of 12 patients (75.0%) had reduction from baseline in target lesion SOD (Figure 1A). Median duration of response was not reached (not reached [NR]; 95% CI, 12.5 months-NR); responses lasted ≥24 months and were ongoing in 4 (66.7%) of the 6 responders (Figure 1B, Supplementary Table S3). Median overall survival (OS) was 19.4 months (95% CI, 7.9 months-NR). Estimated OS rate was 100% at 6 months, 72.7% at 12 months, and 45.5% at 24 months (Supplementary Figure S2). Median progression-free survival (PFS) was NR (95% CI, 1.4 months-NR), with progression-free rates at 12 and 24 months of 64.8% and 51.9% (Supplementary Figure S3). Lifileucel efficacy in this small cohort of patients with MM appeared favorable compared with other therapies in patients with MM. For example, first-line anti-PD-1 or anti-cytotoxic T-lymphocyte antigen-4 (CTLA-4) therapy alone or anti-PD-1 plus anti-CTLA-4 combination therapy was associated with response rates of 13%-29%, median PFS of 2.8-6.8 months, and median OS 11.3-20.4 months in patients with MM [4-6]. In one study, the response rate was 25.0% with nivolumab and 30.4% with nivolumab plus relatlimab, an anti-lymphocyte-activation gene 3 (LAG-3) combination therapy [8]. In a study of patients with MM refractory to anti-PD-1 therapy treated with chemotherapy, the ORR was 10.7% [9]. Notably, the patients in the current study received lifileucel as a later line of therapy compared with the first-line ICI studies [4-6], with 8 of the 12 patients in the current study progressing after first-line ipilimumab plus nivolumab.

The safety profile of lifileucel in patients with MM was consistent with the known safety profile of nonmyeloablative lymphodepletion and IL-2 [7]. All 12 patients experienced ≥1 treatment-emergent adverse event (TEAE; any grade). No grade 5 adverse events were reported. The most common grade 3/4 nonhematologic TEAEs were febrile neutropenia (58.3%) and hypotension (33.3%) (Supplementary Table S4). All 12 patients had grade 3/4 hematologic laboratory abnormalities that recovered to grade ≤2 by day 30 post-lifileucel infusion in most patients (100% for neutropenia, leukopenia, and anemia; 91.7% for thrombocytopenia; 83.3% for lymphopenia and cytopenia).

To compare tumor mutational burden (TMB) in MM and CM, we analyzed DNA from tumor tissue samples (Supplementary Material and Methods). Mean TMB was numerically lower in patients with MM (2.14 mutations/Mb; n = 4) versus CM (10.47 mutations/Mb; n = 47; Figure 1C), but the sample was too small to determine statistical significance. Only 1 of 4 MM samples had an ultraviolet radiation-related Catalogue of Somatic Mutations in Cancer (COSMIC) mutational signature 7 (i.e., SBS7; associated with CC > TT dinucleotide mutations at dipyrimidines), compared with 42 of 47 CM samples (Figure 1D). The limited effectiveness of standard immunotherapies in MM may be due to a lower TMB and other factors, such as differing tumor immune microenvironments in MM versus CM, but here the ORR with lifileucel for MM (50.0%) was higher than for the overall melanoma population (31.4%) [7, 10].

We assessed in vivo persistence of T cells by monitoring the presence of TIL product-specific T-cell receptor (TCR) β chain CDR3 sequences in blood samples (Supplementary Material and Methods). The persistence of TCR clonotypes through month 12 was similar in patients with MM and CM (Figure 1E), suggesting that tumor type does not affect the composition of circulating T cells post-lifileucel infusion. No association was found between persistence and ongoing response, consistent with previous observations [11].

Given the rarity of MM, the sample size for this subgroup analysis was small, limiting generalizability of the findings. Additional study is warranted to confirm response rates in larger cohorts and to better understand the molecular and immunological underpinnings of response.

In conclusion, these results show that lifileucel has clinically meaningful activity with durable responses and no new safety signals in patients with difficult-to-treat MM with progression after anti-PD-1/PD-L1 therapy. This subpopulation analysis further supports the benefit of lifileucel as a one-time treatment that is differentiated from other immunotherapies for melanoma.

Conception and design: Friedrich Graf Finckenstein, Brian Gastman, Jeffrey Chou, Xiao Wu, Giri Sulur, and Rongsu Qi. Financial support: Friedrich Graf Finckenstein, Brian Gastman, Jeffrey Chou, Xiao Wu, Giri Sulur, Rana Fiaz, and Rongsu Qi. Administrative support: Friedrich Graf Finckenstein, Brian Gastman, Jeffrey Chou, Xiao Wu, Giri Sulur, Rana Fiaz, and Rongsu Qi. Provision of study materials or patients: Harriet Kluger, Götz Ulrich Grigoleit, Sajeve Thomas, Evidio Domingo-Musibay, Jason A Chesney, Miguel F Sanmamed, Theresa Medina, Mirjana Ziemer, Eric Whitman, and Amod A Sarnaik. Collection and assembly of data: Friedrich Graf Finckenstein, Brian Gastman, Jeffrey Chou, Xiao Wu, Giri Sulur, and Rongsu Qi. Data analysis and interpretation: Friedrich Graf Finckenstein, Brian Gastman, Jeffrey Chou, Xiao Wu, Giri Sulur, Rana Fiaz, and Rongsu Qi. Manuscript writing: All authors. Final approval of manuscript: All authors.

Harriet Kluger: research funding from institution (Apexigen, BMS, and Merck) and consulting/advisory role (BMS, Chemocentryx, Signatero, Merck, Gigagen, GI Reviewers, Pliant Therapeutics, Eisai, Invox, Wherewolf, Teva, and Iovance Biotherapeutics).

Sajeve Thomas: speaker's bureau (BMS, Merck, Pfizer, Ipsen, Amgen, Genentech, and Foundation One), travel, accommodations, and expenses (BMS, Merck, Pfizer, Ipsen, Amgen, Genentech, and Foundation One), consulting/advisory role (BMS, Merck, Pfizer, Ipsen, Amgen, Genentech, and Foundation One) and research funding (BMS, Merck, Pfizer, Ipsen, Amgen, Genentech, and Foundation One).

Evidio Domingo-Musibay: grants or contracts (Instil Bio).

Miguel F Sanmamed: invited speaker (MSD, BMS, and Roche), advisory board (Numab, BMS, and MSD) and research grant (Roche and BMS).

Theresa Medina: consulting/advisory role (Merck, BMS, Iovance Biotherapeutics, Moderna, Nektar, Regeneron, Exicure, Checkmate, BioAtla, Xencor, Replimune, Day One Pharmaceutical, Pfizer, and Taiga).

Mirjana Ziemer: invited speaker (MSD, BMS, Sanofi, Sunpharma, Pierre Fabre, and Astra Zeneca), advisory board (BMS and Philogen), research grant (Novartis), consulting/advisory role (MSD, BMS, Sanofi, and Sunpharma), and travel, accommodations, and expenses (Pierre Fabre and Sunpharma).

Eric Whitman: consulting/advisory role (Merck) and speaker's bureau (Merck, BMS, Regeneron, and Castle BioSciences).

Friedrich Graf Finckenstein: employment (Iovance Biotherapeutics), stock or stock options (Iovance Biotherapeutics, BMS, and Roche), travel, accommodations, and expenses (Iovance Biotherapeutics), and patents, royalties, and other intellectual properties (Iovance and BMS).

Brian Gastman: employment (Iovance Biotherapeutics), stock or stock options (Iovance Biotherapeutics), and travel, accommodations, and expenses (Iovance Biotherapeutics).

Jeffrey Chou: employment (Iovance Biotherapeutics), stock or stock options (Iovance Biotherapeutics), and travel, accommodations, and expenses (Iovance Biotherapeutics).

Xiao Wu: employment (Iovance Biotherapeutics), stock or stock options (Iovance Biotherapeutics), and travel, accommodations, and expenses (Iovance Biotherapeutics).

Giri Sulur: employment (Iovance Biotherapeutics), stock or stock options (Iovance Biotherapeutics), and travel, accommodations, and expenses (Iovance Biotherapeutics).

Rana Fiaz: employment (Iovance Biotherapeutics), stock or stock options (Iovance Biotherapeutics), and travel, accommodations, and expenses (Iovance Biotherapeutics).

Rongsu Qi: employment (Iovance Biotherapeutics), stock or stock options (Iovance Biotherapeutics), and travel, accommodations, and expenses (Iovance Biotherapeutics).

Amod A Sarnaik: royalties and licenses (Iovance Biotherapeutics), consulting fees (Iovance Biotherapeutics, Guidepoint, Defined Health, Boxer Capital, Huron Consulting Group, KeyQuest Health, Istari, Rising Tide, Second City Science, Market Access, and Gerson-Lehrman Group), honoraria (Society for Immunotherapy of Cancer, Physician's Education Resource, Medscape, WebMD, and Medstar Health), travel, accommodations, and expenses (Iovance Biotherapeutics, and Provectus Biopharmaceuticals), patents (Moffit Cancer Center and Provectus Biopharmaceuticals), and receipt of equipment, materials, drugs, medical writing, gifts, or other services (BMS and Genentech).

This study was sponsored by Iovance Biotherapeutics, Inc. (San Carlos, CA, USA).

This study was approved by Providence Health & Services IRB (PDX15-117); Atlantic Health System IRB (763583); Advarra IRB (PRE00076076); Yale University IRB #2, 3, 4B, 5, 6—Human Investigation Committee IB Oncology, II, III, IV, 1A (1506016045); Western IRB (20160198); University of Louisville IRB (16.0817 [initial approval reference ID: 748942]); Mount Sinai Medical Center IRB (16-49-H-09 [Federalwide Assurance #FWA00000179]); NYU School of Medicine IRB (i16-00804); Sutter Health IRB—(SHIRB) (2016.124 [IRB Net # 931782]); Orlando Health IRB #1 (1092019 [reference: 17.069.060]); Human Research Protection Program, University of Minnesota (STUDY00001236); UC San Diego Human Research Protections Program (171801); Thomas Jefferson University Institutional Review Board (17C.598); Fred Hutchinson Cancer Research Center IRB (9925); Roswell Park Cancer Institute IRB (STUDY00000465/P54117); Medical College of Wisconsin/ Froedtert Hospital IRB—Human Research Protection Program (PRO00031199); Office of the Human Research Protection Program (OHRPP) (IRB#19-000538); University of Miami Human Subject Research Office (HSRO)—IRB (IRB ID: 20190926); CEIm HM Hospitals (Tracked w EudraCT# - 2017-000760-15); Clinical Pharmacological Ethics Committee of the Health Science Council (OGYEI/47943-5/2017); Committee for the Protection of Persons (CPP) South-West and Overseas III (Tracked w EudraCT#- 2017-000760-15); London—West London & GTAC Research Ethics Committee (EudraCT#- 2017-000760-15; MHRA: 48580; IRAS: 229812; REC: 17/LO/1471); Ethics Commission of the Faculty of Medicine at the Technical University of Munich (Tracked w EudraCT#- 2017-000760-15); and Cantonal Commission on Research Ethics involving Human Beings (CER-VD) (2017-02031). All patients provided written informed consent.