Tumor-Infiltrating Lymphocyte Therapy in Melanoma: Facts to the Future

Abstract

Adoptive cell therapy with tumor-infiltrating lymphocytes (TIL) is gaining momentum and demonstrating durable responses in patients with advanced melanoma. Although increasingly considered as a treatment option for select patients with melanoma, TIL therapy is not yet approved by any regulatory agency. Pioneering studies with first-generation TIL therapy, undertaken before the advent of modern melanoma therapeutics, demonstrated clinical efficacy and remarkable long-term overall survival, reaching beyond 20 months for responding patients. TIL therapy is a multistep process of harvesting patient-specific tumor-resident T cells from tumors, ex vivo T-cell expansion, and re-infusion into the same patient after a lymphodepleting preparative regimen, with subsequent supportive IL2 administration. Objective response rates between 30% and 50% have consistently been observed in heavily pretreated patients with metastatic melanoma, including those who have progressed after modern immune checkpoint inhibitors and BRAF targeted agents, a population with high unmet medical need. Although significant strides have been made in modern TIL therapeutics, refinement strategies to optimize patient selection, enhance TIL production, and improve efficacy are being explored. Here, we review past and present experience, current challenges, practical considerations, and future aspirations in the evolution of TIL therapy for the treatment of melanoma as well as other solid tumors.

Introduction

In the last decade, the melanoma treatment landscape has dramatically transformed with approvals for immune checkpoint inhibitor (ICI) antibodies targeting cytotoxic T-lymphocyte antigen-4 (CTLA-4) and programmed cell death receptor-1 (PD-1), as well as small-molecule inhibitors targeting the B-Raf proto-oncogene (1, 2). These treatment modalities improve long-term survival of patients and reduce the risk of relapse in the adjuvant setting (3). Unfortunately, despite these advances, most patients experience disease progression (4). As such, there remains a substantial unmet need to identify effective therapies for treatment-refractory disease.

Adoptive cell therapy (ACT) with tumor-infiltrating lymphocytes (TIL) has emerged as an alternative immunotherapy strategy in melanoma that focuses on harnessing the antitumor abilities of tumor-resident antigen-specific T cells (5–7). The highest overall response rate (ORR, 49%) among patients with advanced melanoma after failure of an approved front-line therapy has been demonstrated with TIL therapy (8). Single-center studies have been critical in laying the foundation for TIL therapy in advanced melanoma (9). Historically, these trials were undertaken in specialized institutions with on-site cell therapy manufacturing facilities (10, 11). Recently, with the expansion of cell therapy technologies, commercial enterprises have established off-site manufacturing facilities that have widened access to TIL therapy with the potential to benefit more patients (12). Accumulated data generated from multicenter, international programs support commercial approval of TIL therapy for the treatment of advanced melanoma, shifting this intervention from an academic pursuit offered at a few select centers to a viable therapeutic option available internationally. Here, we discuss clinical experiences with TIL therapy, the rationale to support future combination with ICIs and other agents, practical considerations for an approved TIL product, and the next generation of therapeutic TILs.

Clinical Experiences

Intrinsic and acquired resistance to front-line ICIs is common and driven by multiple factors (13). Resistance, with a high likelihood of recurrent disease in patients receiving adjuvant ICIs, further emphasizes the need for effective treatments in this ICI-refractory population (14). A variety of immune-activating adoptive cell therapy approaches are being tested in the hope of overcoming these resistance mechanisms (15, 16).

TIL therapy requires harvest of autologous T cells from tumor material by surgical resection, stimulation ex vivo by coculture with cytokines and expansion, to generate the TIL infusion product (15). TILs are infused back into the same patient after non-myeloablative lymphodepletion (NMA-LD) with cyclophosphamide and fludarabine, enabling preferential engraftment of the TIL population (17). Post-TIL infusion, IL2 is administered to facilitate in vivo expansion of the infused T cells (18), followed by a short recovery period to support resolution of IL2-related toxicities (Fig. 1; ref. 19).

Figure 1.

Overview of TIL therapy. Current TIL therapy begins with physician consultation, patient identification, and tumor procurement through surgical resection (step 1). Tumors are then transported centrally to a manufacturing facility or processing hub. TILs are manufactured beginning with digestion and culture or fragmentation of tumors, which yields a suspension of T cells, followed by stimulation and rapid expansion. Manufacturing of unmodified TIL is relatively quick (3 weeks) compared with modified or neoantigen-enriched TIL, which could take several months. The final TIL product is cryopreserved (step 2). TILs are held for later use or immediately transferred to the treatment site. The patient receives NMA-LD for 5 to 7 days, followed by a one-time intravenous infusion of TILs, and supportive IL2 therapy for 3 to 5 days (step 3 A, B, C). The patient is discharged following resolution of chemotherapy and IL2-related toxicity before being evaluated for response (step 4).

Pioneering work in this field was performed by Rosenberg and colleagues beginning in the 1980s, demonstrating the patient-specific antitumor activity of cultured TILs in vivo (20), with clinical effectiveness in diverse cancers (21). Because of the first signal of clinical responses (22, 23), a series of single-center phase I to III clinical trials have demonstrated reproducible efficacy (ORRs, 34%–56%; PFS, 3.7–7.5 months; OS, 15.9–21.8 months) of TIL therapy in metastatic melanoma despite differences in patient characteristics, lymphodepletion regimens, IL2 administration, TIL manufacturing process, and TIL dose (refs. 8, 10, 11, 19, 24–27; Table 1). These encouraging results have stimulated centers worldwide to conduct studies which define the role of TIL therapy in melanoma management and have been summarized previously (15, 16). Here, we focus on the studies that have shifted from an academic single center institution to a centralized manufacturing process to enable broad access to TIL therapy through registrational studies with the intent for regulatory approval.

Table 1.

Clinical efficacy of select TIL therapy trials in melanoma.

Trial	No. of prior therapies, %	Intervention/Pts, n/ disease stage	Lymphodepletion/ IL2 regimen	TIL preparation	TIL product/ IL2 doses received	Efficacy outcomes
Rosenberg et al. 1994 (127)	Surgery, 80% IO, 43% Chemotherapy, 20% Radiotherapy, 10% None, 3% Hormonal therapy, 1%	TIL + IL2±Cy 86 Metastatic disease	Lymphodepletion: ○Cy: 25 mg/kg IL2: ○720,000 IU/kg every 8 hr max 15 doses	Minced into 3- to 5-mm fragments and digested overnight in an enzyme medium	TIL: ○>1011 cells in cycle 1 (85% of pts) ○>2 × 1011 cells in cycle 1 (48% of pts)	ORR: ○IL2 alone: 35% (n = 29) ○IL2 + Cy: 35% (n = 57) ○All: 34% (n = 86)
Schwartzentruber et al. 1994 (128)	Surgery, 95% Chemotherapy, 29% Radiation, 7% Hormonal/non–IL2 IO, 67% IL2-based therapy, 49% TIL therapy, 14%	TIL + IL2 (±IFN-α) ±Cy 43 Stage IV	Lymphodepletion: ○Cy: 25 mg/kg IL2: ○720,000 IU/kg IV every 8 hr OR 216,000 IU/kg and IFN-α: 3 × 106 U/m2 IV every 8 hr	Minced and enzymatically digested Cells were cultured in IL2–containing media for 28 to 84 d	TIL (mean ± SEM): ○Cycle 1: responders: 1.9 ±0.1×10−11; nonresponders: 1.5 ± 0.1×10−11 ○Cycle 1 + 2: responders: 3.2 ± 0.3×10−11; nonresponders: 2.4±0.2×10−11 IL2 doses (mean ± SEM): ○Cycle 1: responders: 2.6 ±0.4 × 10−6 IU/kg; Cycle 1: nonresponders: 2.5 ±0.3 × 10−6 IU/kg ○Cycle 1 + 2: responders: 5.8 ±0.9×10−6 IU/kg; nonresponders: 4.9 ± 0.5×10−6	ORR: 21%
Dudley et al. 2002 (23)	IL2, 100% Chemotherapy, 62%	TIL+ Cy/Flu + IL2 13 HLA-A2+ metastatic melanoma	Lymphodepletion: ○Cy: 60 mg/kg for 2 d + Flu 25 mg/ m2 for 5 d IL2: ○720,000 IU/kg every 8 hr until tolerable toxicity	Small (2 mm3) tumor fragments or enzymatically digested tumor tissue TIL cultures screened for autologous tumor cell and HLA-A2+ reactivity Expanded to ≥5×107 cells (3–6 weeks)	TIL (average, range): ○7.8×1010 (2.3×1010 – 13.7×1010) IL2 (average, range): ○9 (5 – 12)	ORR: 46% (n = 6)
Dudley et al. 2010 (129)	IL2, 62%	Lymphodepletion±TBI + TIL + HD IL2 56 M1a-M1c	Lymphodepletion: ○Cohort 1: Cy 60 mg/kg for 2 d + Flu 25 mg/ m2 for 5 d (n = 33) ○Cohort 2: Cy 60 mg/kg for 2 d + Flu 25 mg/ m2 for 5 d (n = 33) + TBI 3×2 Gy (n = 23)	CD8+ enriched young TILa	TILb (average± standard error): ○Cohort 1: 47.7 ±3.3×109 ○Cohort 2: 43.1 ± 7.5×109 IL2: ○Cohort 1: 6.3 ± 0.3 ○Cohort 2: 7.5 ± 0.5	ORR: ○Cohort 1: 58% ○Cohort 2: 48% ○All: 54%
Rosenberg et al. 2011 (9)	Median = 2 (mean ± SEM = 2 ±0.2) IL2, 83% IFN, 56% Chemotherapy, 43% IL2+Chemotherapy, 40% Anti–CTLA-4, 12% IL2+anti–CTLA-4, 9% IL2+anti–CTLA-4 + chemotherapy, 7% None, 5%	Lymphodepletion±TBI + TIL + HD IL2 93 M1a-M1c	Lymphodepletion: ○Cohort 1: Cy 60 mg/kg for 2 d + Flu 25 mg/ m2 for 5 d (n = 43) ○Cohort 2: Cy 60 mg/kg for 2 d + Flu 25 mg/ m2 for 5 d + TBI 2 Gy (n = 25) ○Cohort 3: Cy 60 mg/kg for 2 d + Flu 25 mg/ m2 for 5 d +12 TBI (TBI 2 × 2 Gy/d for 3 d) (n = 25) IL2: ○720,000 IU/kg every 8 hr until tolerable toxicity, max 15 doses ○Cohort 2/3 received ≤2 × 106/kg of CD34+ HPSCs	Single-cell suspensions or 1–2 mm3 fragments in 24-well cultures plates Lesion size >2 cm in diameter	TIL (mean ± SEM): ○CR (n = 20): 6.5 ± 0.7 × 10−10 ○PR (n = 32): 6.1 ± 0.5 × 10−10 ○NR (n = 41): 5.5 ± 0.6 × 10−10 IL2 (mean ± SEM): ○CR: 7.1 ± 0.4 ○PR: 7.7 ± 0.5 ○NR: 8.8 ± 0.4	ORR: ○Cohort 1: 49% (n = 43); (CR, 12%) ○Cohort 2: 52% (n = 25); (CR, 20%) ○Cohort 3: 72% (n = 25); (CR, 40%) ○All: 56% (n = 93) 3-yr OS rate: ○CR: 100% ○PR: 31% ○NR: 7% ○All: 36% 5-yr OS rate: ○CR: 93% ○PR: 21% ○NR: 5% ○All: 29%
Radvanyi et al. 2012 (130)	Chemotherapy, 29% Biochemotherapy, 29% IO, 39% Unknown, 3%	Lymphodepletion + TIL + 2 cycles HD IL2 31 Stage IIIc, IV	Lymphodepletion: ○Cy 60 mg/kg for 2 d (on d −7 and −6) + Flu 25 mg/m2 for 5 d from d −5 to −1 IL2: ○1st dose: 720,000 IU/kg until tolerable toxicity, max 15 doses; 2nd dose: 21 d post TIL	1 lesion used in most cases, in some cases two to three smaller nodules 3 to 5 mm3 cut fragments; manual dissection; pre-REP TIL cryopreserved (5 wk), thawed, further expanded to post-REP TIL Treatment: post-REP TIL	TIL (range): ○8 to 150×109 IL2: ○2 cycles (n = 28/31 pts)	mOS: NR mPFS: 7.6 mo (95% CI, 4.1 mo -22.2 mo)
Besser et al. 2013 (38)	Previous therapy, 100% IL2-based therapy, 95% Ipilimumab therapy, 23%	Lymphodepletion + TIL + HD IL2 80 Stage IV (M1a-M1b) BRAFV600/E mutations (n = 34/80)	Lymphodepletion: ○Cy 60 mg/kg for 2 d + Flu 25 mg/ m2 for 5 d IL2: ○720,000 IU/kg until tolerable toxicity, max 15 doses	Fragmentation, enzymatic digestion, and cell remnants Young-TIL Pre-REP cultures (2 wk), cryopreserved or expanded (14 d) Treatment: young TIL (n = 57)	TIL (total cell number, n = 57): 52 ± 24 × 109	ORR: ○40% (n = 57); (CR, 6%; PR, 23%) ○29% (n = 80); (CR, 9%; PR, 32%) Treated pts (n = 57) ○mOS: 15.2 mo ○mPFS: 4 mo 1-yr PFS rate: 42% 1-yr OS rate: 50% 2-yr OS rate: 45% 3-yr OS rate: 42%
Anderson et al. 2016 (25)	IL2-based, 96% Ipilimumab-based, 80% Responders: median 2 (range, 1–4) Nonresponders: median 2 (range, 2–4)	Lymphodepletion + TIL + LD IL2 25 Stage IIIc, IV	Lymphodepletion: ○Cy 60 mg/kg for 2 d (d −7 and −6) + Flu 25 mg/m2 for 5d (d −5 to −1) IL2: ○Decrescendo regimen (18 MIU/m2 for 6 hr, 18 MIU/ m2 for 12 hr, 18 MIU/ m2 for 24 hr followed by 4.5 MIU/ m2 for another 3×24 hr) (prescheduled IL2 dose was reduced by 25%–50% in 4 pts)	Lesions ≤1 cm3 were surgically resected and cut into 2 to 3 mm3 fragments Pre-REP/ young TIL in 21 d (range, 13–36) were cryopreserved or entered REP	TIL (median): ○98 × 109 (range 61–20 × 109) IL2 (median): ○112 MIU dose	ORR: 42% (n = 25) mPFS: All: 3.9 mo mOS: All: 21.8 mo 1-yr OS rate: 72% 3-yr OS rate: 40.8%
Goff et al. 2016 (24)	None, 26% HD IL2, 29% Anti–CTLA-4, 31% Anti–PD-1, 3% Anti–CTLA-4 and anti–PD-1, 8% Adjuvant (IFNα, vaccine, etc.), 38% Chemotherapy, 11% BRAF and/or MEK inhibitor, 9% Other (including biochemotherapy), 10%	NMA-lymphodepletion ±TBI + TIL + HD IL2 101c Advanced disease (76%, M1c)	Lymphodepletion: ○Arm 1: NMA, Cy 60 mg/kg for 2 d + Flu 25 mg/m2 for 5 d ○Arm 2: Cy 60 mg/kg for 2 d + Flu 25 mg/m2 for 5 d + TBI 2 Gy 2×/d (total of 1200 TBI) IL2: ○720,000 IU/kg every 8 hr until tolerable Cohort 2 received ≤2×106/kg of CD34+ HPSCs	Single fragments of 2 to 3 mm2 were enzymatically digested and cryopreserved Autologous reactive or actively growing cells were cryopreserved and/or selected for REP	IL2 median (range): ○Arm 1: 6 (4–7) ○Arm 2: 5 (3.3–6)	ORR: 54% (n = 54) ○Arm 1: 45% (n = 23); (CR, 24%; PR, 22%) ○Arm 2: 62% (n = 31); (CR, 24%; PR, 38%) mPFS ○Arm 1: 7.5 mo ○Arm 2: 9.6 mo mOS ○Arm 1: 36.6 mo ○Arm 2: 38.2 mo
van den Berg et al. 2020 (26)	Chemotherapy, 70% Anti–CTLA-4, 100% Adjuvant (IFNα, vaccine, etc.), 40% BRAF and/or MEK inhibitor, 40%	NMA-lymphodepletion + TIL + HD IL2 10 Stage IIIc/ IV	Lymphodepletion: ○Cy 60 mg/kg for 2 d + Flu 25 mg/ m2 for 5 d IL2: ○600,000 IU/kg every 8 hr until tolerable toxicity, max 15 doses	Enzymatic digestion Growth 2–4 weeks to reach ≥1×108 cells followed by cryopreservation or REP TILs harvested additional 14 days after growth	TIL (range): ○1.7 – 19.6 × 1010 IL2 median (range): ○3.5 (1–9)	ORR: 50% (n = 5) ○CR, 20% ○PR, 30%
Sarnaik et al. 2021 (12)	Mean = 3.3 (range, 1–9) Anti–PD-1 or PD-L1, 100% Anti–CTLA-4, 80% Anti–PD-1+ CTLA-4, 52% BRAF ± MEK, 88% IL2, 11% Surgery, 99% Radiotherapy, 52%	Lymphodepletion + TIL + HD IL2 66 Stage IIIc, IV	Lymphodepletion: ○Cy 60 mg/kg for 2 d + Flu 25 mg/m2 for 5 d IL2: ○600,000 IU/kg every 8 to 12 hr for up to 6 doses, starting within 3 to 24 hr after TIL infusion	At least one resectable lesion (or aggregate of lesions) measuring a minimum of 1.5 cm in diameter postresection Mean target lesion diameter = 106 mm Centralized 22-d process and cryopreserved product	TIL administered: ○Total: >1 × 109 – <150 × 109 ○Mean: 27.3 × 109 (range, 1.2 × 109–99.5 × 109) IL2: ○Median 5.5 (range 1–6)	ORR: 36% (n = 24) ○(CR, n = 2; PR, n = 22) DCR: 80% (n = 53) mOS: 17.4 mo (95% CI, 11.0-Not reached) 1-yr OS rate: 58% (95% CI, 45%-69%)
Seitter et al. 2021 (10)	≥1 prior systemic therapy, 83% Anti–CTLA-4, 30% Anti–PD-1+, 12% Anti–PD-1+ CTLA-4, 4% BRAF±MEK, 8% IL2, 46%	Lymphodepletion + TIL + HD IL2 226 Metastatic melanoma	Lymphodepletion: ○Cy 60 mg/kg for 2 d + Flu 25 mg/ m2 for 5 d IL2: ○720,000 IU/kg every 8 hr until tolerable	Multiple fragments of 2 to 3 mm2 placed into a single well of a 24-well tissue culture plate, supplemented with media with HD IL2 followed by REP or cryopreserved	TIL administered: ○Maximum: 2 × 1011 IL2: ○Median 6 (range 5–8)	ORR: 51% (n = 116) ○(CR, n = 49; PR, n = 67) mOS: 20.6 mo (95% CI, 15.2–29.9) 3-yr OS rate: 41% 5-yr OS rate: 35% 10-yr OS rate: 32%
Levi et al. 2022 (33)	Anti–PD-1+, 38% Anti–CTLA-4, 31% Anti–PD-1+ CTLA-4, 93% BRAF±MEK, 13% HD IL2, 31% IFNα, 30% Chemotherapy, 14%	Lymphodepletion ± TBI + TIL + HD IL2 ± pembrolizumab 181 Metastatic melanoma	Lymphodepletion: ○Arm 1: Cy 60 mg/kg for 2 d + Flu 25 mg/m2 for 5 d ○Arm 2: Cy 60 mg/kg for 2 d + Flu 25 mg/m2 for 5 d + 1,200 TBI cGy for 3 d ○Arm 3: Cy 60 mg/kg for 2 d + Flu 25 mg/m2 for 5 d + 2 mg/kg pembrolizumab every 3 weeks for total of 4 doses IL2: ○720,000 IU/kg every 8 hr until tolerable	At least one resectable lesion measuring ≥1 cm REP with irradiated PBMC feeder cells, anti-CD3 antibody and IL2	TIL administered: ○Total not reported IL2: ○Median not reported	ORR: ○Anti–PD-1 naïve: 55% (CR, 51%) ○Prior anti–PD-1 therapy: 26% (CR, 8%)
Haanen et al. 2022 (8)	None, 11% Anti–PD-1, 86%	Lymphodepletion + TIL + HD IL2 OR ipilimumab 168 (84 per arm) Unresectable stage IIIc to IV melanoma	Lymphodepletion: ○Cy 60 mg/kg/d for 2 d + Flu 25 mg/m2/d for 5 d IL2: ○600,000 IU/kg every 8 hr	Tumor digested and/or fragmentated for initial outgrowth REP with anti-CD3 antibody, feeder cells, and IL2	TIL administered: ○Total: 5×109 – 2×1011 IL2: ○Median not reported	ORR: ○TIL: 49% (CR, 20%) ○Ipi: 21% (CR, 7%) mPFS: ○TIL: 7.2 mo ○Ipi: 3.1 mo mOS: ○TIL: 25.8 mo ○Ipi: 18.9 mo 6-mo PFS rate: ○TIL: 53% ○Ipi: 21% 2-yr OS rate: ○TIL: 54% ○Ipi: 44%

Abbreviations: BRAF, B-Raf proto-oncogene, serine/threonine kinase; CD, cluster of differentiation; CR, complete response; Cy, cyclophosphamide; d, day; Flu, fludarabine; HD, high-dose; hr, hour; IO, immunotherapy; LD, low-dose; MEK, mitogen-activated protein kinase kinase; mo, months; NE, not estimable; NR, nonresponder; OS, overall survival; s.c., subcutaneous; PBMC, peripheral blood mononuclear cell; PFS, progression-free survival; PR, partial response; pts, patients; RFS, relapse-free survival; TBI, total body irradiation; wk, week, yr, year.

^aIn the cohorts treated in this report, there was no difference between the age of CD8⁺ enriched young TIL cultures for responding and nonresponding patients.

^bThree additional patients received cryopreserved TIL from prior resections.

^cOf 101 patients randomly assigned to cohorts 1 or 2, all completed their planned treatment course except two patients in cohort 2 (TBI arm), whose treatment was aborted for progressive disease.

In today's modern era of TIL therapy, studies have incorporated efficiencies in production, treatment, and product characterization. Recently, lifileucel (LN-144), comprising autologous, unmodified TIL infusion, has demonstrated encouraging results in patients with unresectable stage III and IV melanoma in the post–PD-1 inhibitor setting (NCT02360579; ref. 12; Table 1). In this multicenter, international, single-arm phase II multicohort study, TILs were unmodified and centrally manufactured in approximately 3 weeks (12). In cohort 2, nearly all 66 patients had progressive disease despite prior anti–PD(L)-1 exposure; the mean number of prior therapies was 3.3; 17 patients were B-Raf^V600 mutation–positive, of whom 88% had progressed on BRAF targeted agents; 34 patients previously received combination anti–PD-1/anti–CTLA-4, either as front-line therapy (23%) or after failing front-line therapy (29%; ref. 12). For all patients, the ORR was 36% (mOS, 17.4 months), with two complete responses (CR) and 22 partial responses. The best ORR was observed in patients with progressive disease after initial anti–PD(L)-1 therapy (n = 42, ORR = 41%) and was consistent after anti–lymphocyte-activation gene (LAG) 3-containing regimens (12, 28). Recent results from the pivotal cohort 4 of the study (n = 87) demonstrated an ORR of 29% (29). When analyzed together, cohorts 2 and 4 led to a combined ORR of 31%, providing an effective and durable (12-month response rate, 54%) treatment option for heavily pretreated patients with PD-1 refractory melanoma (29). Collectively, lifileucel demonstrated meaningful responses in advanced melanoma and may become the first TIL therapy approved in this setting.

A retrospective analysis of an independent single-site compassionate use program with an alternative unmodified TIL product currently under commercial development reported a response rate of 67% in 21 patients (mOS 21.3 months); the median number of prior therapies was 2 (30). For the 12 patients who received prior anti–PD-1 and anti–CTLA-4 therapy, the response rate was 58% (30). Although responses were rigorously collected with RECIST-compatible assessments in the majority of patients, retrospective analyses of overall response rates in nontrial settings do have limitations. A large-scale, international phase II study (NCT05050006) is currently evaluating an updated version of the manufacturing process for that product, ITIL-168, in patients with advanced melanoma relapsed or refractory to anti–PD-1 therapy (31). Hannen and colleagues reported the first multicenter phase III study of TIL therapy (NCT02278887) where patients with unresectable stage IIIC to IV melanoma were randomized to receive TIL therapy (n = 84) or ipilimumab (n = 84; ref. 8). The majority of patients (86%) were refractory to anti–PD-1 therapy. TIL therapy resulted in improved PFS (median, 7.2 months vs. 3.1 months, respectively), ORR (49% vs. 21%), and OS (median, 25.8 months vs. 18.9 months) compared with ipilimumab (Table 1). Taken together, these results, and the aforementioned findings, support a new era of cell-based personalized therapeutics, demonstrating feasibility of TIL therapy with consistent response rates and opportunities for paradigm-shifting approaches to treating melanoma.

Despite new standards of care (32), retrospective analyses suggest that PD-1 experienced patients may be less responsive to TIL therapy than PD-1 naive patients (10, 33). Among 112 anti–PD-1 naive and 69 anti–PD-1 experienced patients with metastatic melanoma who responded to TIL therapy, those with T cells that recognized at least one tumor neoantigen demonstrated response rates of 56% (CR, 51%) and 41% (CR, 8%), respectively (33). In another retrospective analysis of the experience at the NCI, Seitter and colleagues demonstrated response rates of 56% (mPFS 6.5 months) in PD-1 naive patients and 24% (mPFS 3.2 months) in PD-1 refractory patients (all patients, mOS 20.6 months; ref. 10). Prospective trials are needed to confirm these differences. Given the higher response rates in PD-1 naive patients (Table 1), TIL therapy administered earlier in the treatment sequence may provide a greater benefit. It is unknown whether patients who progress after first-line anti-PD-1 inhibition should consider TIL as a second-line option rather than proceeding immediately to combination ipilimumab/nivolumab therapy. Cumulatively, efficacy of TIL therapy in the setting of anti–PD-1 refractory disease compares favorably to other immunotherapy options which may provide a lower ORR (range, 13%–31%) and shorter PFS (range, 2–5 months; refs. 30, 34–37). Although initial indications suggest that anti–PD-1, anti–LAG-3, and anti–CTLA-4-refractory tumors respond to TIL therapy (9, 28, 38), this question will need to be supported with further research, such as differences in T-cell functionality (39).

Adverse events

Administration of TIL requires trained, experienced staff and access to intensive care unit support (40, 41). The most common toxicities during TIL therapy are attributable to NMA-LD and/or IL2 (15, 16, 41, 42). Expected treatment-emergent adverse events (AE) from NMA-LD include grade 3 or 4 cytopenias in most patients (12, 16), whereas toxicities associated with IL2 are dose- and schedule-dependent (25). IL2-related toxicities are generally predictable, manageable, and transient (11, 42, 43). Administration of supportive high-dose IL2 therapy is medically challenging, and the optimal IL2 dose and schedule is as yet undefined (16, 44). The lifileucel regimen incorporates intravenous bolus IL2 (600,000 IU/kg) every 8 to 12 hours for up to six doses (12), whereas other protocols are utilizing lower doses of IL2 (Table 2). Even so, the T-cell supportive role that IL2 plays in TIL therapy, rather than a directly therapeutic intent, allows for the avoidance of severe IL2-related toxicities through the discontinuation of IL2 at the earliest sign of significant toxicity (12, 42). TIL infusion itself is generally uneventful (41). Rarely, administration can be associated with dyspnea, chills, and fever in the brief period following infusion (15). These AEs have not been correlated with high serum levels of circulating cytokines and should not be confused with cytokine release syndrome commonly reported with other cell therapies like chimeric antigen receptor (CAR) T-cell therapy (45). Given the theoretical risk of TIL-mediated neurologic events from infiltration into the central nervous system and complications of IL2 administration for patients with active brain lesions (46), the safety of TILs in patients with brain metastasis is currently being addressed in clinical trials, although early evidence of CNS response has been seen (47).

Table 2.

Select accruing trials of TILs in solid tumors.

Type of TIL/trial number	Phase/study start date/estimated enrollment, n/sponsor	Tumor type(s)	Select eligibility criteria	Select treatment details	Select primary outcome measures
ITIL168, unmodified TIL NCT05393635a	Phase I June 2022 n = 27 Instil Bio, Inc.	CC HNSCC NSCLC	Cohort 1: CC, progressive disease during or after platinum-based chemotherapy; CPS ≥1 with progression during or after CPI Cohort 2: HNSCC, progressive disease during or after platinum-based chemotherapy Cohort 3: NSCLC, progressive disease during or after platinum-based chemotherapy and CPI Symptomatic and/or untreated CNS metastases are excluded	Regimen: ○Lymphodepletion + ITIL-168 + IL2 + pembrolizumab Lymphodepletionb: ○Cy and Flu × 5 d TILb: ○ITIL-168, autologous TIL IL2b: ○≤8 doses Pembrolizumabb: ○1 dose after tumor resection prior to ITIL-168, ≤1 year	Frequency and severity of TEAEs, serious AEs, and AEs of special interest
ITIL-306, modified TIL NCT05397093a	Phase Ia/b June 2022 n = 51 Instil Bio, Inc.	OC NSCLC RCC	Cohort 1: High-grade serous, endometrioid, or clear cell epithelial carcinoma of the ovary, fallopian tube, or peritoneum (phase I only); progressive disease during or after 1 prior line of chemotherapy Cohort 2: Squamous cell carcinoma or adenocarcinoma of the lung (phase I only); progressed after 1 prior line of platinum-based doublet chemotherapy and a CPI Cohort 3: Clear cell or papillary RCC (phase I only); progressed after 1 prior line of antiangiogenic therapy and a PD-1 axis inhibitor Symptomatic and/or untreated CNS metastases are excluded	Regimen: ○Lymphodepletion + ITIL-306 Lymphodepletionb: ○Cy and Flu × 3 d TILb: ○ITIL-306, autologous TIL	Frequency and severity of TEAEs, serious AEs, and AEs of special interest
IOV-4001, modified TIL NCT05361174a	Phase I/II June 2022 n = 53 Iovance Biotherapeutics, Inc.	Melanoma NSCLC	Cohort 1: Stage IIIc, IIId, or IV unresectable or metastatic melanoma; progressed within 12 weeks of anti–PD-1/PD-L1 txt Cohort 2: Stage III or IV NSCLC; ≤3 prior lines Symptomatic untreated brain metastases are excluded	Regimen: ○Cohort 1,2: Lymphodepletion + IOV-4001 + IL2 Lymphodepletionb: ○Cy and Flu TILb: ○IOV-4001	Phase 1: Safety Phase 2: ORR
Lifileucel, unmodified TIL NCT05398640	N/A; expanded access June 2022 Intermediate size population Iovance Biotherapeutics, Inc.	Melanoma	Progressed on or did not achieve a response or were intolerant due to toxicity after one to four prior lines of therapy Uveal or ocular melanoma is excluded	Regimen: ○NMA-LD + lifileucel + IL2 TILb: ○Lifileucel, autologous TIL	N/A
De-TIL-0255, modified TIL (131) NCT05107739	Phase I December 2021 n = 54 Nurix Therapeutics, Inc.	PROC EC CC	Recurrent or persistent platinum-resistant epithelial ovarian cancer (EOC), including primary peritoneal and fallopian tube carcinoma Recurrent, metastatic, or persistent cervical carcinoma with progression after treatment with taxane-containing regimen Advanced or recurrent endometrial cancer after ≥2 prior lines of therapy Known untreated brain metastases are excluded	Regimen: ○De-TIL-0255 TIL: ○De-TIL-0255 ○DLT period: 1×109 - 150×109, modified TIL	Safety Tolerability Preliminary antitumor activity
ITIL168, unmodified TIL (31) NCT05050006	Phase II October 2021 n = 130 Instil Bio, Inc.	Melanoma	Cohort 1: R/R to ≥1 prior line including a PD-1iCohort 2: Intolerant to a PD-1i and have persistent disease after PD-1i Cohort 3: Stable disease after ≤4 doses of a PD-1i Noncutaneous melanoma and symptomatic/ untreated CNS metastasis are excluded	Regimen: ○Lymphodepletion + ITIL-168 + IL2 Lymphodepletionb: ○Cy and Flu × 5 d TIL: ○5×109 autologous TIL IL2b: ○HD, ≤ 8 doses	ORR
TIL, unmodified NCT05098184	Phase I September 2021 n = 50 Shanghai Gencells Therapeutics Co., Ltd.	Melanoma	Primary, relapsed (failed standard txt or lacks standard regimens), or metastasized melanoma	Regimen: ○Lymphodepletion + TIL Lymphodepletionb: ○Cy and Flu TIL: ○1×109 - 5×1010 autologous TIL	AE ORR DCR DOR PFS OS
MASE-T cells, modified NCT04904185	Phase I August 2021 n = 12 National Center for Cancer Immune Therapy (CCIT-DK)	Melanoma	HLA-A2 positive melanoma Progressive disease on or after anti–PD-1/anti–PD-L1 ± anti–CTLA-4 therapy CNS metastases are excluded	Regimen: ○Arm A: Lymphodepletion+ MASE-T ○Arm B: Lymphodepletion+ MASE-T + pembrolizumab Lymphodepletion: ○Cy (500 mg/m2/day) and Flu (30 mg/m2/day) T cellsb: ○Antigen specific, ex vivo expanded T cells from peripheral blood T cells Pembrolizumab: ○2 mg/kg on day −1 and day +21	Tolerability of the treatment Number of patients excluded due to feasibility issues Number of patients excluded due to feasibility issues
LN-145, unmodified TIL NCT04614103	Phase II May 2021 n = 95 Iovance Biotherapeutics, Inc.	NSCLC	PD-L1 negative (TPS <1%) prior to their CPI treatment (cohort 1) PD-L1 positive (TPS ≥1%) prior to their CPI treatment (cohort 2) PD-L1 negative (TPS <1%) and unable to undergo surgical harvest for TIL generation (cohort 3) Retreatment (cohort 4) CNS metastases excluded	Regimen: ○NMA-LD + LN-145 + LD IL2 Lymphodepletion: ○Cy (60 mg/kg × 2 doses) + Flu (25 mg/m2 × 5 doses) TILb: ○LN-145, autologous TIL IL2: ○600,000 IU/kg, ≤6 doses	ORR
RPTR-168, modified TIL NCT04762225	Phase 1/2 May 2021 n = 24 Repertoire Immune Medicines	Melanoma HNSCC CC	Melanoma ○R/R or metastatic disease ○Patients with noncutaneous melanoma must have PRAME+ tumor HNSCC ○Relapsed/refractory to anti–PD-1/anti–PD-L and/or cetuximab ○HPV-16 E6/E7 positivity CC ○Relapsed/refractory to platinum-based chemo or anti–PD-1/anti–PD-L1 ○HPV-16 E6/E7 positivity Active CNS disease and/or carcinomatous meningitis are excluded	Regimen: ○RPTR-168 T cellsb: ○Autologous multitargeted T cells	Number of DLT Frequency of dose interruptions
GEN-011, modified T cells (92) NCT04596033	Phase I November 2020 n = 24 Genocea Biosciences, Inc.	Melanoma NSCLC SCCHN Urothelial carcinoma RCC SCLC CSCC ASCC	Received, been intolerant of, or been ineligible to receive standard-of-care treatment regimen Sufficient stimulatory neoantigens identified in ATLAS	Regimen: ○Cohort 1: Multiple LD of GEN-011 + IL2 ○Cohort 2: Lymphodepletion + single HD of GEN-011 + IL2 Lymphodepletionb: ○Cy + Flu T cellsb: ○≤5 doses without lymphodepletion	Incidence of treatment-related AEs
TIL, unmodified NCT04165967	Phase 1 September 2020 n = 9 University Hospital, Basel, Switzerland, GMP network of Basel	Melanoma	Measurable unresectable or metastatic melanoma with ≥ 1 PD-1 targeted IO Noncutaneous melanoma and uncontrolled CNS metastasis are excluded	Regimen: ○Lymphodepletion + TIL + LD IL2 + nivolumab Lymphodepletionb: ○Cy + Flu TIL: ○5×109 - 2×1011 TIL IL2: ○125,000 IU/kg/d ≤10d Nivolumab: ○3 mg/kg i.v. 1q2 starts 2 wk before TIL	Number of AEs, body temperature, blood pressure, heartbeat, respiratory frequency, and full blood counts
CISH CRISPR TIL, modified TIL NCT04426669	Phase 1/2 May 2020 n = 20 Intima Bioscience, Inc. Masonic Cancer Center, University of Minnesota	GI cancer	Metastatic progressive gastrointestinal epithelial cancer with progressive disease following ≥1 line standard therapy Patients with ≤3 brain metastases that are less than 1 cm in diameter and asymptomatic are eligible	Regimen: ○NMA-LD + CISH CRISPR TIL + IL2 Lymphodepletion: ○Cy (60 mg/kg) + Flu (25 mg/m2) TILb: ○CISH-inactivated autologous TIL IL2: ○720,000 IU/kg/d ≤6 doses	MTD Efficacy Incidence of AEs
TILT-123, oncolytic adenovirus (132, 133) NCT04217473	Phase I February 2020 n = 15 TILT Biotherapeutics, Ltd.	Melanoma	R/R (stage III/IV) melanoma ≥1 prior line of treatment Treated brain metastases that have not progressed in 3 mo before screening are allowed	Regimen: ○6 doses of TILT-123 prior to, during, and after TIL administration TILb: ○Autologous TIL	AEs (serious, nonserious) prior to TIL administration
ATL001, modified T cells NCT03997474	Phase I/II August 2019 n = 40 Achilles Therapeutics	Melanoma	Metastatic or recurrent melanoma with prior PD-1/PD-L1i CNS metastases excluded	Regimen: ○Cohort A: Lymphodepletion +ATL001 + LD-IL2 ○Cohort B: Lymphodepletion + ATL001/ICI + LD-IL2 ○Cohort C: Lymphodepletion + ATL001 + HD-IL2 T cellsb: ○Autologous clonal neoantigen reactive T cells (cNeT)	Treatment-emergent adverse events and serious AEs to evaluate safety and tolerability
ATL001, modified T cells (134) NCT04032847	Phase I/II July 2019 n = 40 Achilles Therapeutics	NSCLC	Advanced NSCLC with prior PD-1/PD-L1i CNS metastases excluded	Regimen: ○Cohort A: NMA-LD + ATL001 + IL2 ○Cohort B: NMA-LD + ATL001 + checkpoint inhibitor Lymphodepletionb: ○Cy + Flu T cellsb: ○Autologous clonal neoantigen reactive T cells (cNeT) IL2b: ○≤10 doses Pembrolizumabb: ○Prior to and after ATL001, ≤12 mo ○Cohort B only	Safety Tolerability
Lifileucel (LN-144) or LN-145, unmodified TIL (53) NCT03645928	Phase II May 2019 n = 135 Iovance Biotherapeutics, Inc.	Melanoma HNSCC NSCLC	Unresectable or metastatic melanoma (stage IIIC or IV) with ≤3 prior lines excluding CPIs (cohort 1A) or previous anti–PD-1 therapy (cohorts 1B,C) Uveal/ocular melanoma are excluded	Regimen: ○Cohort 1A: NMA-LD+ lifileucel + IL2+ pembrolizumab ○Cohort 1B/3B: NMA-LD + LN-145 + IL2 ○Cohort 1C: NMA-LD+ lifileucel + IL2 ○Cohort 2A/3A: NMA-LD + LN-145 + IL2 + pembrolizumab ○Cohort 3C: NMA-LD+ LN-145+ IL2+ ipilimumab + nivolumab Lymphodepletion: ○Cy (60 mg/kg × 2 doses) + Flu (25 mg/m2 × 5 doses) TIL: ○1×109 - 150×109, autologous TIL IL2: ○≤6 doses 600,000 IU/kg Pembrolizumab: ○400 mg, once after resection and continued after IL2 for ≤24 mo (cohorts 1A/2A/3A) Ipilimumab: ○Once prior to resection Nivolumab: ○Once prior to resection, prior to TIL administration, and up to 24 mo (cohort 3C)	ORR and safety profile measured by grade ≥3 TEAEs
RPTR-147, modified T cell (91) NCT03815682	Phase I/II October 2018 n = 240 Repertoire Immune Medicines Merck Sharp & Dohme, LLC	Solid tumors Lymphomas HPV-16 positive tumors	Relapsed/refractory metastatic or locally-advanced solid tumor Patient must have documented HLA-typing results that meet the study requirements Arm B: PD-1 progression Arm C: HPV-16 positive tumors	Regimen: ○Arm A: RPTR-147:1 ○Arm B: RPTR-147:1+ pembrolizumab ○Arm C: RPTR-147:2 TILb: ○Autologous anti-TAA associated antigen T cells loaded with an IL15-Fc nanogel	Number of DLT Frequency of dose interruptions
TIL, unmodified (135) NCT03638375	Phase I/II July 2018 n = 34 Leiden University Medical Center Bristol-Myers Squibb	Melanoma	Stage III or IV melanoma; must have failed on standard txt options Progressive disease on prior txt Stable (≥2 mo) brain metastases with no prior dexamethasone txt are allowed	Regimen: ○Arm A: TIL + nivolumab ○Arm B: TIL + nivolumab + IFNα TIL: ○2.5 × 108 to 7.5 × 108 TIL Nivolumab: ○3 mg/kg i.v. prior to TIL txt PEG-IFNα: ○1 μg/kg/week ≤90 μg/week	Incidence of treatment-related serious AEs
TIL, unmodified NCT03467516	Phase II May 2018 n = 47 UPMC Hillman Cancer Center	Melanoma	Measurable metastatic uveal melanoma and co-enrollment in companion protocol ≤3 brain metastases that are <1 cm are eligible	Regimen: ○Lymphodepletion + TIL + HD IL2 Lymphodepletionb: ○Cy + Flu TIL: ○1×109 - 2×1011 TIL IL2: ○600,000 IU/kg every 8 hr ≤6 doses	ORR
TIL, unmodified NCT03374839	Phase I/II February 2018 n = 11 Nantes University Hospital Bristol-Myers Squibb	Melanoma	Patients with stage IIIb, IIIc, or IV melanoma ≤2 lines of prior txt Brain or bone metastasis and ocular melanoma are not eligible	Regimen: ○TIL + IL2 + nivolumab TIL: ○Phase 1: 5 × 108 TIL, 2 doses ○Phase 2: 1 to 2 × 108 TIL, 2 doses IL2: ○6,00,000 IU/d ×5 d Nivolumab: ○3 mg/kg every 2 wk ≤52 wk	Incidence of TEAEs
LN-145, unmodified TIL (53) NCT03108495	Phase II June 2017 n = 138 Iovance Biotherapeutics, Inc.	CC	Recurrent, metastatic, or persistent squamous cell carcinoma, adenosquamous carcinoma, or adenocarcinoma of the cervix that is not amenable to curative treatment with surgery and/or radiation therapy Cohort 1 and Cohort 2: progression during or following 1–3 prior systemic chemotherapeutic treatments Cohort 2: Previously received treatment with an ICI Cohort 3: Only received prior chemoradiation or surgery for loco-regional disease	Regimen: ○NMA-LD + LN-145 + LD IL2 + pembrolizumab Lymphodepletion: ○Cy (60 mg/kg × 2 doses) + Flu (25 mg/m2 × 5 doses) TIL: ○1 × 109 to 150 × 109, autologous TIL IL2: ○≤6 doses 600,000 IU/kg Pembrolizumab: ○200 mg, once after resection and continued after IL2 for ≤24 mo	ORR AEs Efficacy
T cells, unmodified NCT02133196	Phase II October 2014 n = 85 National Cancer Institute (NCI)	NSCLC	Measurable metastatic (stage IV) or unresectable NSCLC (including but not limited to squamous cell carcinoma, adenosquamous carcinoma, or adenocarcinomas) ≥1 appropriate 1 L systemic therapy and progressed Neuroendocrine tumors are not eligible Patients with ≤3 brain metastases that are ≤1 cm in diameter and asymptomatic are eligible	Regimen: ○Arm 1: NMA-LD+ young TIL + HD aldesleukin ○Arm 2: NMA-LD+ young TIL + LD aldesleukin Lymphodepletion: ○Cy (60 mg/kg/d) + Flu (25 mg/m2/d) TIL: ○Young TILb Aldesleukin: ○Arm 1: 720,000 IU/kg ≤ 9 doses ○Arm 2: 72,000 IU/kg ≤ 12 doses	Response rate
TIL, unmodified (136) NCT02278887	Phase III September 2014 n = 168 The Netherlands Cancer Institute	Melanoma	Unresectable stage III/IV melanoma Received ≤1 prior line, excluding ipilimumab Noncutaneous melanoma, adjuvant ipilimumab within 6 mo from randomization, >2 CNS lesions are excluded	Regimen: ○NMA-LD + TIL + HD IL2 ○Ipilimumab Lymphodepletion: ○Cy (60 mg/kg/d × 2d) + Flu (50 mg/m2 × 5d) TIL: ○5×109 - 2×1011 TIL IL2: ○600,000 IU/kg/d every 8h ≤15 doses Ipilimumab: ○3 mg/kg IV every 3 wk ≤4 doses	PFS at 6 months
TIL + DC vaccine (137) NCT01946373	Phase I October 2013 n = 15 Karolinska University Hospital	Melanoma	Stage III or IV malignant, progressive, melanoma Uveal melanoma included Active CNS metastases are excluded	Regimen: ○Cohort A: NMA-LD + T cells + IL2 ○Cohort B: NMA-LD + T cells + IL2 + DC vaccinations Lymphodepletion: ○Cy (60 mg/kg/d × 2 d) + Flu (25 mg/m2 × 5 d) TIL: ○≤5×1010 TIL IL2: ○100,000 IU/kg as IV bolus over 15-minute period every 8h ≤14 doses DC: ○5 injections with up to 1.5×107 of DC pulsed with autologous tumor lysate and NY-ESO-1 peptide	Safety

Abbreviations: 1L, first-line; ALK, anaplastic lymphoma kinase; ASCC, anal squamous cell carcinoma; ATLAS, Antigen Lead Acquisition System; BRAF, B-raf proto-oncogene, serine/threonine kinase; CNS, central nervous system; CPI, checkpoint inhibitor; CSCC, cutaneous squamous cell carcinoma; Cy, cyclophosphamide; d, day; DC, dendritic cell; DCR, disease control rate; DLT, dose-limiting toxicities; DOR, duration of response; ECGs, electrocardiograms; EC, endometrial cancer; Flu, fludarabine; GI, gastrointestinal; HD, high-dose; HLA, human leukocyte antigens; HNSCC, head and neck squamous cell carcinoma; HPV, human papillomavirus; ICI, immune checkpoint inhibitor; i.d., intradermal; IO, immunotherapy; IV, intravenous; LD, low-dose; MASE, multiple antigen specific endogenously derived; NY-ESO-1, New York Esophageal Squamous Cell Carcinoma-1; ORR, objective response rate; OS, overall survival; PARPi, poly-ADP ribose polymerase inhibitor; PD-1, programmed cell death protein-1; PD-L1, programmed cell death protein-ligand 1; PFS, progression-free survival; PROC, platinum-resistant ovarian cancer; q, every; RCC, renal cell carcinoma; ROS, c-ROS oncogene 1; R/R, relapsed/refractory; sc, subcutaneous; SCCHN, squamous cell carcinoma of the head and neck; SCLC, small cell lung cancer; TEAE, treatment-related adverse events; TKI, tyrosine kinase inhibitor; TPS, tumor proportion score; txt, treatment; UC, urothelial carcinoma; US, United States; wk, week; yr, year.

Trial identification and information compiled from ClinicalTrials.gov (accessed June 3, 2022). Trials are placed in order of the most recent study start date.

^aTrial is open and not yet enrolling.

^bDose level was not specified.

Combining TILs with ICIs

An appealing therapeutic approach to melanoma treatment is the possibility of combining TIL therapy with anti–PD-1 to enhance efficacy and durability of response (48). Evidence indicates that PD-1 regulates the interaction between tumors and autologous T cells, thus, the effects of TIL therapy may be enhanced by blocking immunosuppressive signals in the tumor microenvironment (TME) through checkpoint blockade (49). High PD-1 expression has been observed in tumor-reactive CD8⁺ T-cell subsets post-TIL therapy (50). Tumor-expressed PD-L1 binds to PD-1 on TILs, which counteracts the T-cell receptor (TCR)-signaling cascade and impairs T-cell activation (51). Inhibiting the PD-1/PD-L1 signaling axis is a feasible strategy to restore and reset immune responses in the TME (52). At a median follow-up of 11.5 months, the phase II IOV-COM-202 study (NCT03645928) of combination lifileucel and pembrolizumab in 10 patients with ICI-naive metastatic melanoma recently reported OR and CR rates of 60% and 30%, respectively (53). Although more patients need to be treated before efficacy can be reliably assessed, this combination is feasible without significant increases in toxicity (53). Common grade ≥3 treatment-emergent AEs were manageable and consistent with those expected from pembrolizumab, NMA-LD, and IL2 (53). One downside to this approach is that it shifts TIL therapy from a one-time dosing strategy to require long-term administration of pembrolizumab (≤2 years; ref. 53). Preclinical (54) and ongoing clinical studies (NCT03374839, NCT02621021) suggest that there are multiple mechanisms by which PD-1 blockade may enhance outcomes with ACT, including effects on both the endogenous T-cell response as well as the cell therapy product.

Blockade of CTLA-4 with ipilimumab promotes antigen presenting cell (APC)-mediated T-cell activation and antitumor responses (55). Compared with patients who do not receive anti–CTLA-4, ipilimumab induces broad and frequent T-cell responses against common tumor antigens and may induce naïve-like tumor-infiltrating T cells (56). A small pilot study (NCT01701674) of combination TIL and ipilimumab for n = 13 patients with metastatic melanoma reported an ORR of 38% with median progression-free survival of 7.3 months (57). As new ICIs are being developed, including the recent approval of the LAG-3 inhibitor relatlimab for melanoma (58), opportunities for combination studies with TIL therapy will expand.

Practical Considerations

Patient selection

Selecting patients likely to benefit from TILs is an area of high interest. As with ICIs, patients who appear to benefit most from TILs have slowly progressing soft tissue disease (59). Anecdotally, Mehta and colleagues reported that patients with high disease burden, including disease in sanctuary sites like the brain, have responded to TILs (47), although these patients are typically excluded from TIL trials. Moreover, shorter exposure to prior anti–PD-1 therapy may maximize the duration of response to TIL treatment (60). Prognostic factors, such as serum levels of lactate dehydrogenase, disease burden, and specific organ involvement (e.g., soft tissue disease versus liver, brain, bone metastases), may ultimately be used to identify patients most likely to achieve additional benefit and long-term survival from TIL therapy (4). Emerging biomarkers may predict response to TIL, including checkpoint expression and tumor mutational burden/tumor recognition, which are currently under investigation (61). In light of the recent data that ICIs should be considered before targeted therapy for BRAF-mutated melanoma (62, 63), on-treatment biomarkers [liquid biopsies, circulating tumor DNA (or ctDNA) monitoring, and immune profiling] may help provide the basis for rational treatment sequencing and optimal patient selection (64).

Banking models

One of the largest challenges of TIL therapy is the time needed to harvest and expand the T-cell population, which inevitably generates delays for patient intervention (65). Typical manufacturing times are currently around 3 weeks (12) to 3 months (66). As such, there is great interest in shortening the wait time from clinical decision to treat with TIL until the product is available for infusion. One approach is TIL harvest at an earlier time point prior to when the product might actually be needed (67). TILs might be stored either as the frozen tumor sample or a manufacturing intermediate and the sample will be ready for rapid completion of manufacturing at the time when clinically indicated (68, 69). Best practices and optimal patients to consider for tumor banking have yet to be established (70). The implications of utilizing TIL banked earlier in the patent's course of disease for subsequent treatment of a dynamic and progressing disease remain unknown. Banking may preserve tumor reactive T cells, however the efficacy of TIL products made from banked tumors could be impacted by changes to the patient's tumor during disease progression or intercurrent anticancer therapies. As access to TIL therapy is anticipated to increase in the near future, a comprehensive effort and approach to establish tumor bank initiatives are warranted (71).

Bridging therapy

A strategy to decrease drop-out rates due to disease progression during TIL production includes the administration of “bridging therapies,” allowing systemic therapy to be given between the time of TIL harvest and lymphodepleting chemotherapy (57, 72). Historically, this has not been built into TIL trial protocols, as dropout rates between 12% and 21.9% demonstrate that some patients experienced disease progression that precluded them from receiving TIL after successful harvest and manufacturing (25, 38, 73). However, groups evaluating modified TIL products requiring a protracted manufacturing period of several months are building the option for bridging into their protocols. To mimic expected clinical practice upon approval, the lifileucel expanded access program is allowing bridging therapy for patients with melanoma (NCT05398640) and so will patients with solid tumors receiving ITIL-168 (NCT05393635). For BRAF-mutant melanoma, BRAF-targeted agents are an attractive bridging option, as they have high response rates but with typically limited durability (74). Stopping these agents, even for patients who are experiencing clinical progression, can lead to tumor flare (75), so bridging with BRAF-targeted agents is an appealing option. For BRAF-wild type disease, while driven by prolonged T-cell activation and restored T-cell proliferation needed for T-cell–mediated immunity, CTLA-4 bridging or priming approaches may be limited by the relatively high incidence of long-lasting toxicity (76). A single cycle of cytotoxic chemotherapy may be an alternative, viable option for these patients (77). Furthermore, particularly in BRAF-wild-type disease, a banking approach before embarking on front-line anti–PD-1-based therapy may be preferable.

Cost and centralization of TIL therapy

Costs of manufacturing and administration of TIL is clearly a major concern for healthcare providers and payers (78). It is anticipated that commercial TIL products will have high overhead costs as a result of start-up activities of a TIL therapy program (79). In the context of other therapies that require chronic administration sometimes over years (78), TIL therapy may potentially offer a cost-effective addition to the melanoma armamentarium as it is a personalized, one-time treatment approach (65). With growing experience, delivering some elements of TIL therapy in an out-patient setting may be feasible and reduce high in-patient costs, but will require a multidisciplinary approach and coordination, especially for logistical and reimbursement issues (80). Current models established by approved cell therapies provide the opportunity to leverage and expand upon such successful programs (81).

Other considerations

Clinical and commercial cell therapy products must meet regulatory requirements of safety, purity, and potency prior to manufacturing release. Of the numerous product release assays, potency assays have attracted significant attention due to the complex and/or not fully characterized mechanisms of action for TIL therapy (82). Clinical activity of a TIL product likely depends on several factors including the diversity and functional avidity of the TCR repertoire, the frequency of antitumor clonotypes in the final product, ability of the infused cells to efficiently traffic to tumors, and the phenotypic differentiation and state of exhaustion of antitumor clones (83). Patient-related factors including the status of the endogenous immune system, performance status, and comorbidities, as well as tumor burden and disease sites may well influence response to therapy (6). Furthermore, features of the tumor itself, including dynamic and heterogenous tumor antigen expression (84), complicates the routine assessment of antitumor activity of TIL products since practicalities of tumor procurement may limit capturing the full spectrum of antigens present in the patient (85). Thus, some tumor-specific T-cell clones present in the TIL product may be improperly designated tumor nonreactive. Finally, a potency assay must be robust, rapid, and reproducible within a GMP quality control environment (82). Common components of modern potency assays designed for products made from bulk, unselected TIL include nonspecific, or, less commonly, tumor-specific activation of TIL assessed by multiparametric assays including cytokine expression, upregulation of surface markers of activation and TIL-mediated tumor cell death (86). Although these assays are tuned to describe a TIL product's functionality in vitro, correlation with clinical responses have yet to be confirmed in prospective clinical trials (9, 12, 87, 88). In summary, given these constraints, development of potency assessment to support registrational trials, commercialization, and licensing of these products lags behind the reproducible clinical efficacy of TIL in melanoma, including in multicenter trials designed to lead to regulatory approval.

Future Changes and Technologies

Next-generation TILs

As the field of TIL therapy rapidly evolves, technologic advances are being developed to enhance the antitumor efficacy of the TIL product. The next generation of TILs (Table 3) are based on the pillars of effective immune activation (89). First, TCR recognition of the intratumor clonal neoantigen heterogeneity is a determinant of the adaptive immune response to cancer (Fig. 2, strategy 1; ref. 90). TIL therapies, such as lifileucel and ITIL-168, have shown that an unrestricted TCR repertoire in TIL products can counter tumor heterogeneity and deliver clinical responses (ORRs 36%–67%; refs. 12, 31). Moreover, neoantigen-specific TILs have demonstrated persistence lasting nearly 3 years after TIL infusion (26). Clinical studies of predefined, neoantigen-specific TIL infusion products are underway to create TIL and TCR therapy products with a higher fraction of tumor-specific TCRs (66, 91–94). The possibility to select specific TIL subsets enriched for tumor/neoantigen recognition is a potential strategy to generate specific tumor-reactive TIL (10, 25, 33); trials are ongoing, and data will be forthcoming (Table 2). Phenotypically characterizing the most tumor-reactive cell types to include in TIL products based on surface markers (e.g., PD-1 and CD137; refs. 95, 96) to identify a narrow T-cell subset for TIL therapy use (97) are additional strategies early in development. However, increasing the complexity of the manufacturing process prolongs manufacturing times and risks disease progression and patient withdrawal before accessing treatment (98). The cost–benefit balance to both patients and payers will need to be carefully assessed in prospective studies.

Table 3.

Select list of next-generation therapies targeting T-cell activation.

TIL therapies
Treatment	Category	MOA	Company	Strategy
ATL001 (66)	TIL therapy	Using the VELOS manufacturing process, cNETs that target clonal neoantigens unique to each patient's tumor, and blood sample are manufactured from TILs cocultured with neoantigen peptide-pulsed dendritic cells	Achilles Therapeutics	1
ITIL-168 (31) ITIL-306 (CoStAR) (99, 100, 138)	TIL therapy	ITIL-168: An autologous TIL cell therapy made from digested and cryopreserved tumors; offers an unrestricted TCR repertoire ITIL-306: Novel CoStAR molecule encoding an extracellular FRα-targeting scFv and intracellular CD28 and CD40 signaling sequences transduced into autologous TILs	Instil Bio, Inc.	1 1, 2
CISH CRISPR TIL(bioRxiv 2021.08.17.456714) (139)	TIL therapy	Genetically engineered, neoantigen-specific TIL in which the intracellular immune checkpoint CISH has been inhibited using CRISPR/Cas9-gene editing	Intima Bioscience, Inc.	1
Lifileucel (LN-144) (12) LN-145 (53) IOV-4001 (111, 112)	TIL therapy	Lifileucel: Cryopreserved TIL infusion products LN-145: Cryopreserved TIL infusion products IOV-4001: TALEN-mediated PDCD-1 knockout TIL cell therapy; abrogates the need for anti-PD-1 therapy	Iovance Biotherapeutics, Inc.	1 4 4
PH-762 (110)	TIL therapy	INTASYL-mediated gene silencing of PD-1 in TIL	Phio Pharmaceuticals	4
KSQ-001 (eTIL) (116)	TIL therapy	eTILTM therapy created via CRISPR/Cas9-mediated editing of the novel gene target CT-1, which was identified using a CRISPRomics discovery platform	KSQ Therapeutics	4
DeTIL-0255 (117)	TIL therapy	DeTIL cell therapy derived via ex vivo treatment of autologous-derived TILs with a potent, small-molecule inhibitor of CBL-B known as NX-0255	Nurix Therapeutics, Inc.	4
cytoTIL15 (OBX-115) (102)	TIL therapy	Genetically engineered TILs that express a regulated form of mbIL15 under the control of carbonic-anhydrase-2 DRDs, controlled by the ligand ACZ	Obsidian Therapeutics	3
ADP-TILIL7 (140)	TIL therapy	ADP-TILIL7: Autologous TILs transduced with human IL7 under the control of a NFAT inducible promoter	Adaptimmune Therapeutics	3
TCR-T therapies
SPEAR T-cell therapy (104)	TCR-T therapy	SPEAR T-cell therapy engineered to target MAGE-A4, with the ability to incorporate IL7 and CCL19, which increases migration of SPEAR T cells into tumors	Adaptimmune Therapeutics	1, 3
GEN-011 (92)	TCR-T therapy	NPT that uses ATLAS to identify immunogenic neoantigens in NPTs and excludes Inhibigens, antigen targets of T cells that promote tumor growth	Genocea Biosciences, Inc.	1
NeoTCR-T cells (NeoTCR-P1) (93, 94)	TCR-T therapy	Cancer-specific CD8 T cells derived using the imPACT Isolation Technology platform (potential HLA-binding neoAg in context of the patient's HLA class I haplotype are predicted), TCRs from antigen-experienced CD8 T cells are functionally validated by generating T cells expressing neoTCRs using non-viral precision genome engineering to insert the transgenic TCR chains into the endogenous TRAC locus.	PACT Pharma	1,4
IL12-tethered T cells (RPTR-168) (105, 106) PRIME IL15 (RPTR-147) (91)	TCR-T therapy	RPTR-168: Using the Deep Primed platform, IL12 is tethered to the surface of adoptively transferred T cells RPTR-147: Autologous anti-TAA T cells generated with a proprietary dendritic cell priming process and loaded with an IL15-Fc nanogel; TAAs used included PRAME, NY-ESO-1, SSX2, Survivin, and WT1	Repertoire Immune Medicines	3 3

Abbreviations: ACZ, acetazolamide; ATLAS, antigen lead acquisition system; CCL19, chemokine (C–C motif) ligand 19; CISH, cytokine induced SH2 protein; DRD, drug response domains; DeTIL, drug enhanced TIL; eTIL, engineered TIL; Fc, fragment crystallizable; FRα, folate receptor alpha; MAGE-A4, melanoma-associated antigen-A; mb, membrane bound; neoAg, neoantigens; NFAT, nuclear factor of activated T cells; NPT, neoantigen-targeted autologous peripheral T-cell therapy; NY-ESO-1, New York esophageal squamous cell carcinoma 1; PDCD-1, programmed cell death protein 1; PRAME, preferentially expressed antigen in melanoma; scFv, single-chain fragment variable; TAA, tumor-associated antigens; TCR-T, TCR-engineered T cell; WT1, Wilms tumor 1.

Figure 2.

Strategies to optimize T-cell activation in next-generation TIL. Immune-modulation strategies involve improvements in intracellular and extracellular signaling. Strategy 1. Extracellular T-cell activation occurs via TCR/neoTCR-mediated recognition of TAA or neoAg peptides. Novel therapeutic products select and enrich for pre-existing tumor antigen-specific T cells. Strategy 2. Intracellular and extracellular enhancements of T-cell activation and effector function occur through dual CD28 and CD40 intracellular signaling domain-mediated costimulation upon TCR-mediated antigen recognition. Strategy 3. Extracellular T-cell activation through the local delivery of immunomodulatory molecules such as IL7 and CCL19, as well as cell-anchored IL12, or drug-inducible membrane-bound IL15 expression. Strategy 4. Increasing T-cell fitness and reducing T-cell exhaustion with intracellular strategies such as PDCD-1 knockout with TALEN, and CT-1 knockout with CRISPR/Cas9. CCL, chemokine (C–C motif) ligand; FRα, folate receptor alpha; neoAg, neoantigens; TAA, tumor-associated antigens.

Improved TIL function through synthetic costimulation may mitigate known mechanisms of immune escape and resistance to TIL therapy (Fig. 2, strategy 2; ref. 27). Including a synthetic costimulatory antigen receptor (CoStAR) molecule with dual CD28 and CD40 domains on healthy donor T cells targeting the tumor-associated antigen CEA led to increased T-cell activation and long-term proliferation even in the absence of IL2 (99, 100). Furthermore, the enhancement of cytokine-induced immune activation may be accomplished through the local delivery of cytokines outside of IL2 (Fig. 2, strategy 3; ref. 101). Inclusion of growth-promoting cytokines with TIL products is also being examined. The genetically engineered TIL cytoTIL15 expresses a regulated form of membrane-bound IL15 under the control of carbonic-anhydrase-2 drug response domains, activated by acetazolamide (102). Although the regulated expression of IL15 allows for tunable antigen-independent long-term TIL persistence in a dose-dependent manner (102), secondary effector memory T-cell differentiation may be impaired (103). In addition, T cells with membrane bound IL15 (91), IL7/CCL19 expressing specific peptide enhanced affinity receptor (SPEAR) T cells targeting the tumor antigen MAGE-4 (104), and T cells anchored with IL12 (105, 106) are also under investigation.

Although pitfalls have been encountered when investigating alternative cytokines (107, 108), new therapies may reduce T-cell exhaustion in the TME, which is a defining feature in many cancer types (Fig. 2, strategy 4; ref. 109). To simulate therapeutic effects of PD-1 inhibition, PD-1 silencing with INTASYL-mediated self-delivering siRNA (or PH-762) in TIL products was efficient (85% knockdown), and TILs displayed an activated and improved effector phenotype (110). When using transcription activator-like effector nucleases (TALEN)-based gene knockout of the PD-1 gene PDCD-1 in TILs (IOV-4001), T cells exhibited improved in vivo effector function (111, 112); these results prompted clinical investigation in metastatic melanoma (NCT05361174).

Furthermore, clustered regularly interspaced short palindromic repeats (CRISPR)-based knockout of the negative TCR-signaling regulator cytokine-induced SH2 (CISH) protein in TIL led to increased TCR avidity, neoantigen recognition, and tumor cytolysis (bioRxiv 2021.08.17.456714; refs. 113–115). KSQ-001, an engineered TIL therapy, was created via CRISPR/Cas9-mediated editing of a novel, and yet undisclosed, intracellular immune checkpoint (cell therapy-1, CT-1) identified in peripheral T cells (116), resulting in polyfunctional and proliferative T cells, which are under clinical development (NCT04426669). Ex vivo treatment of autologous TILs with NX-0255, a potent, small-molecule inhibitor of the E3 ubiquitin ligase Casitas B-lineage lymphoma proto-oncogene-B (CBL-B), resulted in drug enhanced TILs (DeTIL-0255) that were less exhaustive and showed enhanced cytolytic T-cell activity (117), which is being tested in gynecologic malignancies (NCT05107739).

Application beyond melanoma

TIL therapy, with its broad and patient-specific polyclonality (15, 26), has exciting potential to overcome clonal tumor heterogeneity and induce deep and durable remissions in a growing list of treatment-refractory cancers other than melanoma (Table 2). In a single-site phase I study, patients with advanced non–small cell lung cancer (NSCLC; including 4 patients with EGFR mutations), who progressed after nivolumab monotherapy received TIL followed by maintenance nivolumab for up to a year and reached an ORR of 23% (118). Twenty-seven patients with cervical carcinoma (CC) who had progressed on standard of care treatments received TIL and achieved an ORR of 44.4% (119). Lifileucel and LN-145, in combination with pembrolizumab, have also shown promise in advanced ICI-naive head and neck squamous cell carcinoma (HNSCC; n = 18, ORR = 38.9%) and advanced untreated CC (n = 14, ORR = 57.1%), respectively (53). The effectiveness of unmodified TILs in renal cell carcinoma (RCC), gynecologic malignancies, and gastrointestinal cancers have shown mixed results, due to difficulties in TIL manufacturing (120, 121), changes in subclonal tumor heterogeneity after prior treatment exposure (122), or a low frequency of tumor reactive TILs (123). In preclinical models, preferential expansion of CD137/PD-1⁺ TIL may select for rare tumor-reactive TIL, thereby making this treatment modality potentially relevant for cancer types that have not previously demonstrated efficacy with nonselected TIL therapies, including myeloma and colorectal cancer (95, 96, 124, 125). Furthermore, enriched neoantigen-specific TILs have shown promise in treatment-refractory breast cancer when combined with pembrolizumab (n = 6, ORR = 50%; ref. 126). Despite these challenges, TILs are currently being explored in these indications and it is hoped that novel approaches such as those summarized in Table 3 may prove beneficial over time.

Conclusions

TIL therapy is a rapidly evolving modality, which is expected to take its place alongside ICI as part of the growing immunotherapy toolkit used to treat melanoma and other cancer types. In melanoma, data are accumulating that demonstrate significant antitumor efficacy with unmodified TILs. Advancements in our understanding of the TME provide the opportunity to enhance the therapeutic window to achieve the true potential of TIL therapy. Numerous paths to this outcome exist, including process improvements, cell engineering, and combinations with other available agents. Encouragingly, several next-generation cellular therapies are currently under or are rapidly headed for clinical evaluation. The optimal timing and sequencing of TIL therapy needed to maximize efficacy will continue to emerge as a focus in anticipation of TIL approvals. Although challenges exist, attention must focus on achieving regulatory approval of TIL therapy in melanoma and further optimizing this approach, including thorough product manufacturing and characterization, to help establish a new option to extend long-term survival in this disease.

Authors' Disclosures

A. Betof Warner reports personal fees from Bristol-Myers Squibb, BluePath Solutions, Instil Bio, Lyell Immunopharma, Immatics, Novartis, and Pfizer and personal fees and other support from Iovance Biotherapeutics outside the submitted work. P.G. Corrie reports other support from Instil Bio and Iovance outside the submitted work. O. Hamid reports personal fees from Instil Bio during the conduct of the study as well as personal fees from Alkermes, Amgen, Beigene, Bioatla, BMS, Eisai, Roche Genentech, Georgiaimune, Giga Gen, Grit Bio, GSK, Idera, Immunocore, Incyte, IO Biotech, Iovance, Janssen, Merck, Moderna, Novartis, Obsidian, Pfizer, Regeneron Sanofi, Seattle Genetics, Tempus, Vial, and Zelluna and personal fees and other support from Bactonix outside the submitted work; in addition, O. Hamid's institution has research contracts with Arcus, Aduro, Akeso, Amgen, Bioatla, BMS, Cytomx, Exelixis, Roche Genentech, GSK, Immunocore, Idera, Inyte, Iovance, Merck, Moderna, Merck Serono, Nextcure, Novartis, Pfizer, Regeneron, Seattle Genetics, Torque, and Zelluna.