Efficacy and safety of one-time autologous tumor-infiltrating lymphocyte cell therapy in patients with recurrent and/or metastatic head and neck squamous cell carcinoma

Abstract

Background

Recurrent and/or metastatic head and neck squamous cell carcinoma (HNSCC) has a high recurrence rate after first-line immunotherapy or chemoimmunotherapy. The presence of a high density of tumor-infiltrating lymphocytes (TILs) in HNSCC tumors was shown to be associated with improved clinical outcomes. One-time autologous TIL cell therapy was evaluated in patients with recurrent and/or metastatic HNSCC.

Methods

C-145-03 (NCT03083873) was a phase 2 study of TIL in patients with recurrent and/or metastatic HNSCC assigned to 1 of 4 treatment cohorts: cohort 1, non-cryopreserved TIL; cohort 2, cryopreserved lifileucel (22-day manufacturing); cohort 3, cryopreserved lifileucel (16-day manufacturing); cohort 4, cryopreserved LN-145-S1 programmed cell death protein-1 (PD-1) selected. Patients underwent tumor resection for TIL generation. After preparative non-myeloablative lymphodepletion, patients received a single infusion of TIL followed by interleukin-2 (IL-2) infusion(s). The primary endpoint was investigator-assessed objective response rate (ORR) per Response Evaluation Criteria for Solid Tumors (RECIST) V.1.1. Secondary endpoints were investigator-assessed duration of response (DOR), disease control rate (DCR), progression-free survival, overall survival, and incidence of treatment-emergent adverse events.

Results

Overall, 53 patients received TIL: cohort 1 (n=8), cohort 2 (n=17), cohort 3 (n=16), cohort 4 (n=12). Median age was 57 years and most patients were males (87%; 46/53) with stage IV disease (98%; 52/53). Patients had a median of two prior lines of systemic therapy; 87% (46/53) of patients had prior anti-PD-1/programmed cell death ligand-1 therapy and 72% (38/53) had prior chemotherapy. The ORR was 11% (6/53) with six patients achieving partial response (cohort 1, n=3; cohort 2, n=1; cohort 4, n=2). At median follow-up of 17.9 months, the median DOR was 7.6 months. The DCR was 76% (40/53); 64% (34/53) of patients had stable disease. The safety profile was consistent with known toxicities associated with non-myeloablative lymphodepletion and IL-2 administration.

Conclusions

This study demonstrated the feasibility of consistently generating sufficient TIL from HNSCC tumors. Results from this study suggest TIL cell therapy may serve as a potential treatment option for patients with HNSCC and support further development, including TIL cell therapy combined with immune checkpoint inhibitors or other agents or with other TIL products.

Trial registration number

NCT03083873.

WHAT IS ALREADY KNOWN ON THIS TOPIC

• A recent real-world study (Black CM, et al Front Oncol. 2023;13:1160144) reported that the most common second-line treatment for patients with recurrent and/or metastatic head and neck squamous cell carcinoma (HNSCC) who received first-line pembrolizumab alone or in combination with chemotherapy was platinum-based regimens (41% and 45%, respectively), followed by other non-immunotherapy regimens (25% and 33%, respectively). Therefore, there is an unmet need for effective alternative immunotherapy options in patients with recurrent and/or metastatic HNSCC whose disease progressed during or after immune checkpoint inhibitor (ICI) therapy. As tumor-infiltrating lymphocyte (TIL) cell therapy demonstrated durable responses in the treatment of unresectable or metastatic melanoma, and the presence of a high density of TIL was associated with improved clinical outcomes in patients with HNSCC, exploration of the potential benefit of autologous TIL cell therapy in HNSCC is warranted.

WHAT THIS STUDY ADDS

• This study demonstrated the feasibility of consistently generating TIL from HNSCC tumors. Notably, we demonstrated that TIL derived from HNSCC tumors may induce partial responses in heavily pretreated patients with recurrent and/or metastatic HNSCC, including those previously treated with ICI and/or chemotherapy who have limited treatment options.

HOW THIS STUDY MIGHT AFFECT RESEARCH, PRACTICE OR POLICY

• Data suggest that TIL cell therapy may be a potential treatment option for patients with recurrent and/or metastatic HNSCC and support further development, including TIL cell therapy combined with ICIs or other agents or with other TIL products.

Background

Head and neck squamous cell carcinoma (HNSCC) comprises a group of malignancies that arise from mucosal epithelial cells lining the oral cavity, pharynx, larynx, and sinonasal tract.¹ In the USA, HNSCC accounts for approximately 3.6% of all cancers with an estimated 71,100 new diagnoses and 16,110 deaths in 2024.²

Treatment for recurrent, unresectable, and/or metastatic HNSCC is determined based on prior treatment, performance status, comorbidities, disease and symptom burden, programmed cell death ligand-1 (PD-L1) expression in tumor and immune cells, microsatellite instability, and human papillomavirus (HPV) status.³ Based on findings from the phase 3 KEYNOTE-048 study, pembrolizumab in combination with platinum and fluorouracil is approved as first-line standard-of-care treatment for patients with recurrent or metastatic HNSCC; pembrolizumab monotherapy is approved as first-line standard-of-care treatment for patients with PD-L1-positive recurrent or metastatic HNSCC.4,6

Nivolumab or pembrolizumab monotherapy demonstrated improved overall survival (OS) in patients without prior immune checkpoint inhibitor (ICI) exposure, or with disease recurrence within 6 months after platinum-based chemotherapy and chemoradiation administered as adjuvant therapy or in the setting of primary or recurrent disease.^{7 8} Patients treated with anti-programmed cell death protein-1 (anti-PD-1) therapy after platinum-based chemotherapy achieved response rates of 13.3–14.6% with median OS of 7.5–8.7 months and median progression-free survival (PFS) of 2 months.^{7 8} The anti-epidermal growth factor receptor antibody cetuximab is also approved for the treatment of metastatic or recurrent HNSCC in combination with platinum-based chemotherapy.⁹ Cetuximab combined with cisplatin administered to patients who had stable or progressive disease (PD) after receiving two cycles of platinum-based doublet chemotherapy achieved objective response rates (ORRs) of 10–20%; median PFS ranged from 2.0 to 4.9 months, and median OS ranged from 4.3 to 11.7 months.¹⁰ The combination of cetuximab plus nivolumab in patients with previously treated or platinum-resistant disease demonstrated response rates of 22%, with median PFS and OS of 3.4 months and 11.5 months, respectively.^{11 12} Treatment with the ErbB inhibitor afatinib after progression on or after platinum-based chemotherapy yielded an ORR of 10%, median PFS of 2.6 months, and median OS of 6.8 months.¹³

There are few effective treatment options in patients who experience disease progression on or after ICI therapy. In light of this unmet need, adoptive T-cell-based therapy may be a promising option.^{14 15} Neoantigen-specific CD8+ T cells derived from various tumor types have been shown to express PD-1.16,18 These PD-1-positive tumor-infiltrating lymphocytes (TILs) demonstrated superior antitumor activity compared with PD-1-negative TILs, and persistence was required to induce a long-term response.^{16 18 19} Evidence suggests that patients with HNSCC who have high levels of TIL are less likely to have disease recurrence than those with low or moderate TIL levels, and that TIL level is an independent predictor of disease-free survival.²⁰ A high density of TIL has been shown to correlate with better OS in patients with oropharyngeal squamous cell carcinoma.^{21 22} Patients with HNSCC who had dense CD3+ and CD8+ T cell infiltration within their tumors had significantly prolonged OS, PFS, and distant metastasis-free survival.²³ Furthermore, levels of PD-1-positive TIL were positively correlated with a favorable clinical outcome in patients with HNSCC; therefore, selection for PD-1-positive TIL is expected to enhance the antitumor effect of TIL.¹⁷ Selection and enrichment for PD-1-positive TIL from HNSCC tumors may enhance their potency and efficacy in the treatment of recurrent and/or metastatic HNSCC.

Adoptive cell therapy with TIL is being evaluated in multiple solid tumor malignancies with melanoma being the most extensively studied tumor type.²⁴ Lifileucel is a one-time autologous T-cell immunotherapy (more commonly referred to as TIL cell therapy) approved by the US Food and Drug Administration for the treatment of adult patients with unresectable or metastatic melanoma previously treated with a PD-1-blocking antibody and, if BRAF V600 mutation positive, a BRAF inhibitor with or without a MEK inhibitor.²⁵

Notably, HNSCC is among the tumor types with the highest immune cell infiltration,²⁶ and tumor-reactive TIL can be successfully isolated and expanded from HNSCC patient tumor samples.²⁷ However, sites of resection require careful selection because of the increased risk of bacterial growth in the oral mucosa and development of fungating wounds.^{28 29} Here, we report findings from C-145-03, a phase 2 open-label study (ClinicalTrials.gov: NCT03083873) evaluating autologous TIL cell therapy in patients with recurrent and/or metastatic HNSCC.

Methods

Study design

C-145-03 was a phase 2 multicenter, multicohort, non-randomized, prospective, open-label study conducted between January 2017 and August 2022 across 18 sites in the USA that evaluated LN-145/LN-145-S1 autologous TIL cell therapy in patients with recurrent and/or metastatic HNSCC. The study was approved by the Institutional Review Board (IRB) or Institutional Ethics Committee (IEC) and conducted in accordance with ethical principles of the Declaration of Helsinki and Good Clinical Practice (GCP) guidelines of the International Conference on Harmonization (ICH) and regulations of the US Food and Drug Administration. The study protocol (online supplemental file 1) and informed consent form were also approved by the study sponsor and the IRB or IEC. All patients provided informed consent in accordance with ICH and GCP guidelines. Patients were sequentially enrolled into 1 of 4 treatment cohorts: cohort 1, non-cryopreserved TIL; cohort 2, cryopreserved TIL (lifileucel) (22-day manufacturing process); cohort 3, lifileucel (16-day manufacturing process); cohort 4, PD-1–selected cryopreserved TIL (LN-145-S1). The treatment period spanned up to 12 days (figure 1) and consisted of non-myeloablative lymphodepletion (up to 7 days), TIL infusion (1 day), and interleukin-2 (IL-2) administration (1–4 days).

Figure 1. CONSORT diagram. Of the 64 patients enrolled, 53 patients received TIL and were included in the full analysis set and safety analysis set. The full analysis set was defined as patients who received TIL that met the manufacturing product specifications. The safety analysis set was defined as patients who received TIL. ^aIndicates PD-1-selected cryopreserved TIL. CONSORT, Consolidated Standards of Reporting Trials; PD-1, programmed cell death protein-1; TIL, tumor-infiltrating lymphocyte.

Patient population

Patients ≥18 years of age with recurrent and/or metastatic HNSCC (based on histologic evaluation of the primary tumor) who received one to three lines of prior systemic immunotherapy and/or chemotherapy for HNSCC were enrolled. Eligible patients included those with ≥1 resectable lesion (yielding ≥1.5 cm diameter of tumor tissue) for TIL generation and measurable disease (as defined by Response Evaluation Criteria for Solid Tumors (RECIST) V.1.1 criteria) following tumor resection for TIL manufacturing. Patients also were required to have an Eastern Cooperative Oncology Group (ECOG) performance status of 0 or 1 and an estimated life expectancy of ≥6 months.

Key exclusion criteria were organ allograft or prior cell transfer therapy, concurrent systemic steroid therapy >10 mg of prednisone or other steroid equivalent, hypersensitivity reactions to any component or excipients of TIL cell therapy or other study drugs, symptomatic and/or untreated brain metastases, and active systemic infections, coagulation disorders, or other major medical cardiovascular, respiratory, or immune disorders. Complete inclusion and exclusion criteria are presented in the online supplemental table 1.

Tumor resection and TIL generation

Tumor tissue for TIL generation was ideally obtained from visceral locations that had not been previously irradiated or if at least 3 months had elapsed between irradiation and resection. Tumor resection sites included primary sites, regional lymph nodes, or distant metastases (skin, abdomen, chest wall, mediastinum, pericardium, liver, lung, distant lymph nodes). Sites to be resected were carefully selected to avoid areas with a high risk of contamination (eg, areas of prior irradiation or ulcerated tumors) or microbial colonization (eg, bowel lesions).³⁰ Resected tumor samples underwent pathological evaluation to ensure the presence of viable tumor tissue. Tumor samples were shipped to the manufacturing site for TIL generation. The 22-day manufacturing process consisted of two steps. In the first step, known as the pre-rapid expansion protocol (REP) step, tumor tissues were divided into fragments and placed in cell culture medium containing a high concentration of IL-2 that allows TIL to migrate from the tissue fragments. In the second step, known as the REP step, migrated TIL are cultured in the presence of IL-2, OKT3 monoclonal antibody, and irradiated peripheral blood mononuclear cells (iPBMC; as feeder cells) for TIL activation and expansion. The 16-day process is a shortened version of the 22-day process, in which the migrating TIL are immediately activated with IL-2, iPBMC, and OKT3. For PD-1-selected TIL, the 22-day manufacturing process included the addition of a PD-1 selection step. Following expansion, TILs were harvested and formulated in transportation media or cryopreservation media and were subsequently shipped to the clinical site for infusion.

Treatment

Preparative non-myeloablative lymphodepletion was scheduled to start 7 days before TIL infusion (online supplemental figure 1). The non-myeloablative lymphodepletion regimen consisted of intravenous cyclophosphamide 60 mg/kg/day for 2 days followed by intravenous fludarabine 25 mg/m²/day for 5 days. A single infusion of TIL (1×10⁹ to 150×10⁹ cells) was administered at least 24 hours after the last infusion of fludarabine. A short course of high-dose IL-2 600,000 IU/kg intravenously was initiated between 3 and 24 hours after completion of TIL infusion; patients received up to 6 IL-2 doses administered every 8–12 hours over 4 days. No bridging therapy was permitted per protocol.

Endpoints and assessments

The primary endpoint of the study was investigator-assessed ORR per RECIST V.1.1 criteria. Secondary endpoints were investigator-assessed duration of response (DOR), investigator-assessed disease control rate (DCR), investigator-assessed PFS, OS, and incidence of treatment-emergent adverse events (TEAEs).

Radiographic disease assessment—via CT, CT positron emission tomography, or MRI—was performed at screening, baseline, every 4 weeks from TIL administration for the first 3 months, at approximately 4.5 and 6 months after TIL infusion, and approximately every 3 months thereafter until disease progression as defined by RECIST V.1.1 criteria or withdrawal of consent by the patient. Presence of measurable disease and response was assessed by the investigator using RECIST V.1.1 criteria. Patients meeting RECIST V.1.1 criteria for a confirmed complete response (CR) or partial response (PR) as assessed by investigator were classified as responders in the analysis of ORR. DOR was defined as the time from first response (CR or PR) to the date of PD; DCR was defined as the percentage of patients who achieved confirmed CR, PR, or stable disease (SD). PFS was defined as the time from the date of TIL infusion to the date of PD, or death due to any cause; OS was defined as the time from date of TIL infusion to the date of death due to any cause. Safety assessments included TEAEs, which were graded using the National Cancer Institute (NCI) Common Terminology Criteria for Adverse Events (CTCAE) V.4.03. TEAEs include all adverse events from the start of TIL infusion and up to 30 days after TIL infusion for all cohorts.

The TIL products were assessed by flow cytometry for T-cell subsets, other immune cells, and phenotypic markers. The TIL products and preinfusion and postinfusion PBMC were sequenced using the iRepertoire iR-Complete assay (Huntsville, Alabama, USA) to identify and quantify T-cell receptor (TCR) clonotypes and their frequency. For each patient, the total proportion of TCR clonotypes in each PBMC sample that was also found in TIL was calculated and used to measure postinfusion T-cell persistence.

Statistical analysis

No formal hypothesis testing was performed. Data were analyzed for each cohort using descriptive statistics; no statistical comparisons were performed. Based on an estimation of ORR using the half width 95% CI of <0.21 (for cohort 2) and <0.23 (for cohorts 3 and 4) by the Clopper-Pearson method, a total of 55 patients were planned for enrollment: 8 patients in cohort 1, 17 patients in cohort 2, up to 15 patients in cohort 3, and up to 15 patients in cohort 4.

Investigator-assessed ORR and DCR were reported as a point estimate with two-sided 95% CI limits based on the Clopper-Pearson exact methods. Patients without any baseline or any post-baseline measurements were considered non-evaluable. Time to event variables of PFS, OS, and DOR were analyzed using Kaplan-Meier estimates and summary statistics. For DOR analyses, patients who did not experience PD or death prior to the time of final database lock were censored at their last radiologic disease assessment before the start of a new anticancer therapy. For PFS analyses, patients who did not experience PD or death at the time of database lock were censored on the last date of tumor assessment. For OS analyses, patients who remain alive at the time of final database lock were censored on the last date of their known survival status. TEAEs were summarized as counts and percentages.

Study populations analyzed included the enrolled set, the safety analysis set, and the full analysis set. The enrolled set was defined as all patients who underwent tumor resection for TIL generation regardless of whether or not they received TIL therapy. The full analysis set was defined as all patients who received TIL that meets the manufacturing specifications. The safety analysis set was defined as patients who received TIL. Results from analyses of TIL products and correlation with response were analyzed using the Wilcoxon rank-sum test.

Results

Patient disposition and baseline characteristics

As of the data cut-off (October 12, 2022), 64 patients were enrolled in the study (enrolled set). Of the 64 patients enrolled, 53 received TIL that met the manufacturing product specifications and were included in the full analysis and safety analysis sets, of whom 8 were assigned to cohort 1, 17 were assigned to cohort 2, 16 were assigned to cohort 3, and 12 were assigned to cohort 4 (figure 1). The majority of reasons for not receiving TIL were patient-related (physician decision, n=4; withdrawal of consent, n=2; declining health and hospice care, n=1; and death, n=1). TIL was not available for three patients.

Median age of the 53 patients who received TIL was 57 years (range, 24–75); most patients (87%; 46/53) were male with a baseline ECOG performance status of 0 or 1 (89%; 47/53), and almost all (98%; 52/53) had stage IV disease at study entry (table 1). The most common types of HNSCC were oropharyngeal (66%; 35/53), oral cavity (13%; 7/53), and supraglottic laryngeal cancer (11%; 6/53). Approximately 51% (27/53) of patients had HPV-positive primary tumors and 62% (33/53) had a PD-L1 combined positive score (CPS) of ≥1 with 34% (18/53) having a PD-L1 CPS ≥20. Less than 6% (3/53) of patients were current tobacco users; 47% (25/53) of patients were former tobacco users, and 47% (25/53) never used tobacco. Patients had received a median of two prior lines of therapy (range, 1–4), and most had received prior anti-PD-1 and PD-L1 immunotherapy (87%; 46/53) or prior chemotherapy (72%; 38/53). The median target lesion sum of diameter was 66.0 mm (range, 14–177). Sites of tumor resection for each cohort are shown in online supplemental table 2.

Table 1. Demographic and baseline characteristics.

Characteristic	Cohort 1 n=8	Cohort 2 n=17	Cohort 3 n=16	Cohort 4 n=12	Total N=53
Median age, years (range)	57 (24–64)	58 (37–71)	56 (34–67)	56.5 (50–75)	57 (24–75)
Sex, n (%)
Female	1 (13)	2 (12)	2 (13)	2 (17)	7 (13)
Male	7 (88)	15 (88)	14 (88)	10 (83)	46 (87)
ECOG performance status, n (%)
0	5 (63)	9 (53)	7 (44)	8 (67)	29 (55)
1	2 (25)	5 (29)	7 (44)	4 (33)	18 (34)
≥2	1 (13)	3 (18)	2 (13)	0	6 (11)
Primary tumor location, n (%)
Oropharynx	4 (50)	12 (70)	11 (69)	8 (67)	35 (66)
Oral cavity	2 (25)	3 (18)	1 (6)	1 (8)	7 (13)
Supraglottic larynx	1 (13)	1 (6)	3 (19)	1 (8)	6 (11)
Nasopharynx	1 (13)	0	0	1 (8)	2 (4)
Maxillary sinus	0	1 (6)	0	1 (8)	2 (4)
Hypopharynx	0	0	1 (6)	0	1 (2)
Disease stage at initial diagnosis, n (%)
0	0	0	0	1 (8)	1 (2)
I	0	2 (12)	0	0	2 (4)
II	1 (13)	1 (6)	2 (13)	1 (8)	5 (9)
III	1 (13)	1 (6)	1 (6)	1 (8)	4 (8)
IV	5 (63)	10 (59)	12 (75)	4 (33)	31 (58)
Unknown	0	2 (12)	1 (6)	5 (42)	8 (15)
Missing	1 (13)	1 (6)	0	0	2 (4)
Distant metastasis at diagnosis, n (%)
M0	4 (50)	10 (59)	13 (81)	10 (83)	37 (70)
M1	3 (38)	5 (29)	3 (19)	1 (8)	12 (23)
Missing	1 (13)	2 (12)	0	1 (8)	4 (8)
HPV status of primary tumor,* n (%)
Positive	3 (38)	10 (59)	10 (63)	4 (33)	27 (51)
Negative	1 (13)	4 (24)	1 (6)	3 (25)	9 (17)
Not assessed	4 (50)	3 (18)	5 (31)	5 (42)	17 (32)
HPV subtype,† n (%)
Subtype 16	2 (67)	10 (100)	8 (80)	4 (100)	24 (89)
Subtype 18	0	0	0	0	0
Subtype 16 and 18	0	0	1 (10)	0	1 (4)
Unknown	1 (33)	0	0	0	1 (4)
Missing	0	0	1 (10)	0	1 (4)
Disease status at screening, n (%)
Metastatic	8 (100)	17 (100)	16 (100)	11 (92)	52 (98)
Recurrent	0	0	0	1 (8)	1 (2)
Stage at study entry, n (%)
Stage IV	8 (100)	16 (94)	16 (100)	12 (100)	52 (98)
Unknown	0	1 (6)	0	0	1 (2)
Median target lesion sum of diameter, mm (range)	78.5 (30–153)	90.0 (44–177)	57.0 (14–163)	55.5 (18–128)	66.0 (14–177)
Median number of target lesions (range)	2.5 (2–4)	3.0 (1–5)	3.0 (1–5)	2.0 (1–4)	3.0 (1–5)
Median number of target and non-target lesions (range)	5.0 (2–10)	4.0 (2–11)	4.5 (2–8)	4.5 (2–8)	5.0 (2–11)
Tobacco use, n (%)
Current	1 (13)	2 (12)	0	0	3 (6)
Former	3 (38)	6 (35)	9 (56)	7 (58)	25 (47)
Never	4 (50)	9 (53)	7 (44)	5 (42)	25 (47)
PD-L1 combined positive score (CPS),‡ n (%)
CPS ≥20	3 (38)	2 (12)	7 (44)	6 (50)	18 (34)
CPS<20	3 (38)	11 (65)	2 (13)	2 (17)	18 (34)
CPS<1	1 (13)	2 (12)	0	0	3 (6)
CPS ≥1	5 (63)	11 (65)	9 (56)	8 (67)	33 (62)
Missing	2 (25)	4 (24)	7 (44)	4 (33)	17 (32)
Median number of lines of prior systemic anticancer therapy (range)	3.0 (1–4)	2.0 (1–3)	2.0 (1–3)	2.0 (1–4)	2.0 (1–4)
Number of lines of prior systemic anticancer therapy,§ n (%)
1	2 (25)	2 (12)	2 (13)	2 (17)	8 (15)
2	0	10 (59)	7 (44)	6 (50)	23 (43)
3	3 (38)	5 (29)	7 (44)	3 (25)	18 (34)
>3	3 (38)	0	0	1 (8)	4 (8)
Prior systemic anticancer therapy modality, n (%)
Chemoradiation therapy	7 (88)	8 (47)	12 (75)	4 (33)	31 (58)
Chemotherapy	6 (75)	12 (71)	10 (63)	10 (83)	38 (72)
Immunotherapy	8 (100)	14 (82)	14 (88)	10 (83)	46 (87)
Radiotherapy	3 (38)	8 (47)	6 (38)	7 (58)	24 (45)
Targeted therapy	7 (88)	9 (53)	9 (56)	6 (50)	31 (58)
Other	2 (25)	4 (24)	6 (38)	3 (25)	15 (28)
Prior systemic anticancer therapy agent category, n (%)
Anti–CTLA-4	2 (25)	3 (18)	1 (6)	0	6 (11)
Anti–PD-1/PD-L1	8 (100)	4 (82)	14 (88)	10 (83)	46 (87)
Monoclonal HER2 antibody	1 (13)	0	0	0	1 (2)
Monoclonal VEGF antibody	0	1 (6)	0	0	1 (2)
Monoclonal anti-EGFR antibody	6 (75)	8 (47)	9 (56)	6 (50)	29 (55)
Targeted TKI	1 (13)	1 (6)	0	0	2 (4)
Median time from last prior anti–PD1/PD-L1 therapy to tumor harvest, months (range)	3.8 (1.1–9.0)	3.3 (1.1–17.6)	2.2 (1.2–10.4)	2.2 (0.00–28.3)	3.1 (0.0–28.3)

^*HPV status at initial diagnosis or historical determination.

^†Percentage is calculated based on number of patients whose HPV status is positive.

^‡PD-L1 CPS score was assessed by a central laboratory.

^§Includes chemoradiation.

CTLA-4, cytotoxic T-lymphocyte associated protein 4; ECOG, Eastern Cooperative Oncology Group; EGFR, epidermal growth factor receptor; HER2, human epidermal growth factor receptor 2; HPV, human papilloma virus; PD-1, programmed cell death protein-1; PD-L1, programmed cell death ligand-1; TKI, tyrosine kinase inhibitor; VEGF, vascular endothelial growth factor.

TIL infusion and IL-2 administration

Overall, patients were treated with a median of 32.4×10⁹ TIL cells (range, 2.4×10⁹ to 98.8×10⁹). Median number of IL-2 infusions was 6 (range, 0–6).

Response outcomes

As of the data cut-off (October 12, 2022), the ORR was 11% (6/53) in the full analysis set; there were six PRs (cohort 1, n=3; cohort 2, n=1; cohort 4, n=2) (table 2; figures2 3); 76% (37/49) of patients had a reduction in tumor burden (figure 2A). The percent change from baseline in target lesion sum of diameters over time by cohort is shown in figure 2B; the response to lifileucel treatment in the one patient with PR in cohort 2 deepened over time.

Table 2. Response outcomes.

Parameter	Cohort 1 n=8	Cohort 2 n=17	Cohort 3 n=16	Cohort 4 n=12	Total N=53
ORR, n (%) (95% CI)	3 (38) (8.5 to 75.5)	1 (6) (0.1 to 28.7)	0	2 (17) (2.1 to 48.4)	6 (11) (4.3 to 23.0)
DCR, n (%) (95% CI)	7 (88) (47.3 to 99.7)	12 (71) (44.0 to 89.7)	12 (75) (47.6 to 92.7)	9 (75) (42.8 to 94.5)	40 (76) (61.7 to 86.2)
Best overall response, n (%)
CR	0	0	0	0	0
PR	3 (38)	1 (6)	0	2 (17)	6 (11)
SD	4 (50)	11 (65)	12 (75)	7 (58)	34 (64)
PD	1 (13)	5 (29)	2 (13)	1 (8)	9 (17)
NE	0	0	2 (13)	2 (17)	4 (8)
DOR of patients with PR, months	2.8* 3.1† 7.6*	23.0*‡	–	7.6* 12.9†‡	2.8* 3.1† 7.6* 7.6* 12.9†‡ 23.0*‡
Median follow-up for response, months	NR	23.0	–	12.9	17.9

^*Patients with known HPV-positive status.

^†Patients with unknown HPV status.

^‡Censored.

CR, complete response; DCR, disease control rate; DOR, duration of response; HPV, human papillomavirus; NE, not evaluable; NR, not reached; ORR, objective response rate; PD, progressive disease; PR, partial response; SD, stable disease.

Figure 2. Best percent change from baseline in target lesion sum of diameters by cohort. (A) Percent change from baseline in target lesion sum of diameters for all patients who received TIL. (B) Percent change from baseline (ie, after tumor resection and before lymphodepletion) over time in target lesion sum of diameters for all patients who received TIL. Across cohorts, a total of 6 patients achieved PR, 32 had SD, and 9 had PD. PD, progressive disease; PR, partial response; SD, stable disease; SOD, sum of diameters; TIL, tumor-infiltrating lymphocyte.

Figure 3. Time to response, duration of response, and time on efficacy assessment after TIL therapy. Across cohorts, a total of six patients achieved a confirmed PR and four patients had a DOR of ≥6 months. DOR, duration of response; PR, partial response; TIL, tumor-infiltrating lymphocyte.

A total of 6 patients were considered responders (figure 3) and 47 patients were classified as non-responders. All responders were male with a median age of 50.5 years (range, 46–62); 85% (40/47) of non-responders were male with a median age of 57 years (range, 24–75). All responders and non-responders had an ECOG performance status of 0 or 1 at screening. A known history of tobacco use was documented in 67% (4/6) of responders and in 51% (24/47) of non-responders; no responders and 6% (3/47) of non-responders were current tobacco users. The primary HNSCC tumor sites in responders were oropharynx (67%; 4/6), oral cavity (17%; 1/6), and supraglottic larynx (17%; 1/6); in non-responders, primary tumor sites were the oropharynx (66%; 31/47), oral cavity (13%; 6/47), supraglottic larynx (11%; 5/47), nasopharynx (4%; 2/47), maxillary sinus (4%; 2/47), and hypopharynx (2%; 1/47). Tumor resection locations for TIL generation in responders were lung (67%; 4/6), neck (17%; 1/6), and lymph node (17%; 1/6); those for non-responders were lung (51%; 24/47), lymph node (17%; 8/47), liver (11%; 5/47), neck (11%, 5/47), oral cavity (2%; 1/47), pharynx (2%; 1/47), or other sites (11%; 5/47). Known HPV-positive primary tumor status was documented in 67% (4/6) of responders (HPV subtype 16, n=3) and in 49% (23/47) of non-responders (subtype 16, n=21; subtype 16 and 18, n=1; subtype missing, n=1); 19% (9/47) of non-responders had known HPV-negative status. Overall, 67% (4/6) of responders and 62% (29/47) of non-responders had a known PD-L1 CPS of ≥1; 33% (2/4) of responders and 34% (16/47) of non-responders had a known PD-L1 CPS ≥20. Of the six responders, 17% (1/6) had received one prior line of therapy, 33% (2/6) had received two prior lines, 17% (1/6) had received three prior lines, and 33% (2/6) had received four prior lines of therapy; all six had received prior anti-PD-1/PD-L1 immunotherapy. Among non-responders, 15% (7/47) had received one prior line of therapy, 45% (21/47) had received two prior lines, 36% (17/47) had received three prior lines, and 4% (2/47) had received four prior lines of therapy; 85% (40/47) had received prior anti-PD-1/PD-L1 immunotherapy.

With a median follow-up for response of 17.9 months (95% CI, 12.9–23.0), the median DOR was 7.6 months (95% CI, 2.8–NR). The individual DORs in the six responders were 2.8, 3.1, and 7.6 months in cohort 1, 23.0+ months in cohort 2, and 7.6 and 12.9+ months in cohort 4. Overall, 64% (34/53) of patients had SD (cohort 1, n=4; cohort 2, n=11; cohort 3, n=12; cohort 4, n=7); the DCR was 76% (40/53). The median duration of SD was 2.8 months (95% CI: 1.9–2.9).

Median PFS was 2.8 months in cohorts 1 and 3 and 1.9 months in cohorts 2 and 4 (online supplemental figure 2). Median OS was 10.4 months in cohort 1, 9.5 months in cohort 2, 9.2 months in cohort 3, and 7.9 months in cohort 4 (online supplemental figure 3).

Safety outcomes

Almost all patients (98%; 52/53) experienced TEAEs (online supplemental table 3). Overall, the most common non-hematologic TEAEs (≥30%) were chills (60%; 32/53), hypotension (53%; 28/53), fever (47%; 25/53), hypophosphatemia (42%; 22/53), febrile neutropenia (42%; 22/53), and hypocalcemia (33%; 18/53). The most common hematologic TEAEs (≥30%) were thrombocytopenia (74%; 39/53), anemia (53%; 28/53), neutropenia (38%; 20/53), and leukopenia (36%; 19/53) (table 3). Frequently observed grade ≥3 TEAEs (≥30%) were thrombocytopenia (62%; 33/53), anemia (45%; 24/53), febrile neutropenia (42%; 22/53), and neutropenia (30%; 16/53). Treatment-emergent hematologic laboratory abnormalities are described in online supplemental tables 4 and 5. Most grade 3/4 hematologic laboratory abnormalities resolved to baseline levels (online supplemental table 5). The incidence of TEAEs of all grades decreased rapidly over the first 2 weeks following TIL cell therapy (online supplemental filgure 4). The TEAE profile was consistent with known safety profiles of non-myeloablative lymphodepletion and IL-2 administration.

Table 3. Treatment-emergent adverse events occurring in ≥30% of patients in any cohort.

Treatment-emergent AEs	Cohort 1 n=8		Cohort 2 n=17		Cohort 3 n=16		Cohort 4 n=12		Total N=53
	Any grade	Grade ≥3	Any grade	Grade ≥3	Any grade	Grade ≥3	Any grade	Grade ≥3	Any grade	Grade ≥3
Non-hematologic AEs, n (%)
Chills	6 (75)	0	12 (70)	0	7 (44)	0	7 (58)	0	32 (60)	0
Hypotension	5 (63)	0	6 (35)	1 (6)	10 (63)	7 (44)	7 (58)	4 (33)	28 (53)	12 (23)
Fever	5 (63)	1 (13)	9 (53)	0	7 (44)	0	4 (33)	0	25 (47)	1 (2)
Hypophosphatemia	4 (50)	3 (38)	6 (35)	3 (18)	9 (56)	5 (31)	3 (25)	1 (8)	22 (42)	12 (23)
Febrile neutropenia	2 (25)	2 (25)	6 (35)	6 (35)	7 (44)	7 (44)	7 (58)	7 (58)	22 (42)	22 (42)
Hypocalcemia	2 (25)	0	5 (29)	2 (12)	8 (50)	5 (31)	3 (25)	0	18 (33)	7 (13)
Hyponatremia	4 (50)	1 (13)	5 (29)	1 (6)	3 (19)	1 (6)	3 (25)	0	15 (28)	3 (6)
Diarrhea	2 (25)	0	4 (24)	0	7 (44)	0	2 (17)	0	15 (28)	0
Hypomagnesemia	1 (13)	0	2 (12)	0	7 (44)	0	5 (42)	0	15 (28)	0
Hypokalemia	3 (38)	1 (13)	4 (24)	0	5 (31)	2 (13)	2 (17)	1 (8)	14 (26)	4 (8)
Dyspnea	2 (25)	0	3 (18)	1 (6)	6 (38)	2 (13)	1 (8)	0	12 (23)	3 (6)
Fatigue	3 (38)	0	1 (6)	0	3 (19)	1 (6)	2 (17)	0	9 (17)	1 (2)
Sinus tachycardia	3 (38)	1 (13)	2 (12)	0	2 (13)	0	1 (8)	0	8 (15)	1 (2)
Cough	2 (25)	0	0	0	5 (31)	0	0	0	7 (13)	0
Peripheral edema	0	0	2 (12)	0	1 (6)	0	4 (33)	0	7 (13)	0
Hematologic AEs,* n (%)
Thrombocytopenia	4 (50)	3 (38)	12 (71)	10 (59)	13 (81)	11 (69)	10 (83)	9 (75)	39 (74)	33 (62)
Anemia	4 (50)	4 (50)	7 (41)	6 (35)	9 (56)	7 (44)	8 (67)	7 (58)	28 (53)	24 (45)
Neutropenia	3 (38)	2 (25)	6 (35)	6 (35)	5 (31)	4 (25)	6 (50)	4 (33)	20 (38)	16 (30)
Leukopenia	3 (38)	2 (25)	6 (35)	5 (29)	5 (31)	4 (25)	5 (42)	4 (33)	19 (36)	15 (28)
Lymphopenia	4 (50)	3 (38)	5 (29)	5 (29)	1 (6)	1 (6)	2 (17)	2 (17)	12 (23)	11 (21)

^*Based on investigator-reported AEs.

AE, adverse event.

Overall, three patients who received TIL experienced grade 5 TEAEs (cohort 3, n=1; cohort 4, n=2). The patient in cohort 3 experienced grade 5 respiratory failure 18 days after lifileucel infusion that was considered related to the lymphodepletion regimen. One patient in cohort 4 experienced grade 5 respiratory failure 7 days after LN-145-S1 infusion that was not related to LN-145-S1, and the other patient in cohort 4 experienced grade 5 respiratory failure 16 days after LN-145-S1 infusion that was considered related to LN-145-S1. No grade 5 events were deemed related to TIL only.

Notably, the incidence of cytokine release syndrome (CRS) was low (4%; 2/53); CRS was reported in two patients (cohort 3, n=1, grade 3; cohort 4, n=1, grade 2) and considered not related to TIL, but related to IL-2. Infusion-related reactions considered related to LN-145-S1 occurred in eight patients (cohort 1, n=1, grade 1; cohort 2, n=2, grade 2; cohort 3, n=4 (n=1, grade 1; n=2, grade 2; n=1, grade 4); cohort 4, n=1, grade 3).

Biomarkers of response

No correlations between the total number of viable cells and immune cell subsets or phenotypic marker expression and treatment response were observed (data not shown). Data related to T-cell persistence were available for two responders, which precluded correlation of T-cell persistence with treatment response.

Discussion

Despite improved survival with anti-PD-1-based ICI therapy,^{5 7 8} only 15–19% of patients with recurrent or metastatic HNSCC in the platinum-refractory and first-line settings remain alive at 4 years.⁶ Few studies have evaluated treatment strategies for patients with recurrent or metastatic HNSCC in the post-ICI setting. While limited findings from non-randomized, small-scale studies suggest that cetuximab in combination with paclitaxel may induce a robust response (ORR), this regimen does not provide a survival benefit.31,34 This phase 2 open-label study of non-cryopreserved, cryopreserved, and PD-1-selected autologous TIL cell therapy resulted in responses in a heavily pretreated recurrent or metastatic HNSCC population, including patients treated with prior lines of ICI and/or chemotherapy. Antitumor responses (ORR, 11%; 6/53) following TIL treatment were observed in a total of six patients (cohort 1, n=3; cohort 2, n=1; cohort 4, n=2) who achieved PR. The DCR was 76% (40/53); 64% (34/53) of patients had SD. With a median follow-up of 17.9 months, all responders had a DOR >6 months except for two patients from cohort 1; the longest response was ongoing at 23.0+ months. Overall, median PFS was 1.9 months, and median OS was 9.5 months. Rates of response, disease control, DOR, and survival outcomes across cohorts in this trial were comparable to those reported in the second-line setting with cetuximab combined with platinum-based chemotherapy,¹⁰ pembrolizumab,¹² nivolumab,¹¹ or afatinib.¹³ Although responses to TIL cell therapy were observed, response rates did not warrant further clinical development of the TIL products explored in this study as therapy for patients with recurrent and/or metastatic HNSCC who have progressed after prior ICIs.

The comparison of efficacy results across cohorts is limited by the small sample size. Although there was a numeric difference in ORRs across treatment cohorts, the ORRs for cohorts 1, 2 and 4 had overlapping 95% CIs. In this study, Cohort 1, which received non-cryopreserved TIL, appeared to have the highest ORR compared with other cohorts that received cryopreserved products. Cohort 3, which received a cryopreserved TIL product derived from a 16-day manufacturing process, did not have responses among the small number of patients evaluated. In a larger C-144–01 study of TIL therapy with lifileucel in patients with advanced melanoma, administration of either non-cryopreserved and cryopreserved TIL resulted in similar ORRs.^{35 36}

The methods of expansion of autologous TIL from excised tumors are well established and sufficiently robust to ensure successful generation of consistently high numbers of high-quality therapeutic cells. Current TIL manufacturing methods span 22 days or less, and the success rate is >90%. As the attributes for release and characterization of TIL (data not shown) were similar regardless of the length of the manufacturing process, it is unlikely that the length of the manufacturing process (22 vs 16 days) contributed to differences in treatment efficacy between Cohort 3 and other cohorts.

Although it is unclear why no responses were observed in patients receiving PD-1–selected cryopreserved TIL, it is important to note that the unique treatment challenges for HNSCC tumors stem partly from their genomic heterogeneity.³⁷ Genomic heterogeneity and instability in tumor cells allow them to evade the immune system via expression of tumor antigens that inhibit the activity of other TCR clonotypes.³⁸ Furthermore, subclonal mutations in tumor cells may modify antigen presentation, making these tumor cells immune-resistant.³⁹ Thus, patients with HNSCC who have a high intratumor heterogeneity have higher mortality than those with low heterogeneity.³⁹

The TIL treatment regimen (cyclophosphamide, fludarabine, TIL cell therapy, IL-2) demonstrated a safety profile that was consistent with the underlying advanced disease and known toxicities associated with non-myeloablative lymphodepletion and IL-2 administration. In the C-145-03 study, a median of 6 (range, 0–6) doses of IL-2 was administered—the same as in the C-144–01 study of lifileucel in patients with advanced melanoma.³⁵ Patients with advanced melanoma in a phase 3 study that compared TIL with ipilimumab received a median of 4 (range, 0–10) doses of IL-2.⁴⁰ Notably, safety outcomes were similar to those observed in the phase 2 C-144-01 study of lifileucel and those in a phase 3 study that compared TIL with ipilimumab in patients with advanced (metastatic or unresectable) melanoma,^{35 40} and were self-limiting in nature. Expected TEAEs with the TIL treatment regimen from the time of lymphodepletion to IL-2 infusion include cytopenias, diarrhea, fever, chills, nausea, and hypotension.^{41 42} The most common grade ≥3 TEAEs observed in the C-145-03 study included thrombocytopenia, anemia, febrile neutropenia, and neutropenia; cytopenias typically resolved within 2 weeks of TIL treatment.

Limitations of the present study include its non-randomized, non-blinded design and small sample size across subsets of different TIL products (cohort 1: non-cryopreserved TIL, n=10; cohort 2: lifileucel (22-day manufacturing), n=22; cohort 3: lifileucel (16-day manufacturing), n=19; cohort 4: LN145-S1, n=13). In the present study, only 13% (7/53) of patients had primary tumors in the oral cavity. The low proportion of oral cavity tumors may stem from the high likelihood of local recurrence and diminished prognosis in patients with advanced disease,^{43 44} which may preclude their eligibility for participation in clinical trials of TIL therapy due to the time taken from tumor harvest to TIL infusion. Furthermore, the increased risk of bacterial contamination associated with oral cavity tumors makes these tumors less preferable for TIL generation. Overall, results from this study demonstrate the feasibility of generating TIL from HNSCC tumors and provide evidence that treatment with TIL can provide antitumor activity in patients with recurrent or metastatic HNSCC whose disease has progressed after prior ICI therapy and/or chemotherapy. Toxicities observed with TIL cell therapy were mostly transient and could be managed by standard supportive measures.

Conclusion

Modest activity following TIL cell therapy was demonstrated in heavily pretreated patients with recurrent and/or metastatic HNSCC; however, response outcomes do not warrant further clinical development of the tested TIL products as monotherapy for patients whose disease has progressed after prior immunotherapy-containing regimens. Resection of tumor tissue for TIL therapy manufacturing was associated with risks typically observed with surgical procedures and administration of anesthesia. All TIL products manufactured for this study met the required specifications, and toxicities were consistent with the one-time administration of the TIL regimen. These results suggest that TIL cell therapy may serve as a potential treatment option for patients with HNSCC and support further development of TIL treatment strategies, including TIL cell therapy combined with other agents, novel TIL products, or TIL products developed using other manufacturing processes.

Data availability statement

All data relevant to the study are included in the article or uploaded as supplementary information.