Therapeutic potential of tumor-infiltrating lymphocytes in non-small cell lung cancer

Abstract

Lung cancer is the leading cause of cancer-related death worldwide, with poor outcomes even for those diagnosed at early stages. Current standard-of-care for most non-small cell lung cancer (NSCLC) patients involves an array of chemotherapy, radiotherapy, immunotherapy, targeted therapy, and surgical resection depending on the stage and location of the cancer. While patient outcomes have certainly improved, advances in highly personalized care remain limited. However, there is growing excitement around harnessing the power of tumor-infiltrating lymphocytes (TILs) through the use of adoptive cell transfer (ACT) therapy. These TILs are naturally occurring, may already recognize tumor-specific antigens, and can have direct anti-cancer effect. In this review, we highlight comparisons of various ACTs, including a brief TIL history, show current advances and successes of TIL therapy in NSCLC, discuss the potential roles for epigenetics in T cell expansion, and highlight challenges and future directions of the field to combat NSCLC in a personalized manner.

1. Introduction to T Cell Therapies

Despite advances in the spectrum of therapies, the 5-year survival rate of lung cancer in the U.S. remains among the lowest of all cancer types at 23% (other sites: pancreas 12%, esophagus 21%, liver 21%) and is the leading cause of cancer related death among both men and women ¹. Lung cancer in general is immensely heterogeneous, first being subcategorized as small cell lung cancer (SCLC), a highly aggressive neuroendocrine tumor, and NSCLC, which includes all other lung carcinomas. In this review, we focus on NSCLC, which accounts for 85% of overall cases and can be further divided into three main subtypes: lung adenocarcinoma (LADC), lung squamous cell carcinoma (LSCC), and mixed/poorly differentiated. The LADC subtype accounts for approximately 45% of diagnoses and is typically found in the distal lung/bronchus, whereas LSCC comprises 18% of cases and often presents proximally^2,3. Adding further to NSCLC heterogeneity is the high mutation rate, with hundreds of somatic mutations per genome - largely attributed to smoking and other environmental exposure^4,5. This magnifies the difficulty for clinicians to identify effective targeted therapies but can also be a vulnerability of cancer due to the high rate of neo-antigen presentation for T cell recognition^6,7.

Metastatic NSCLC harboring actionable driver mutations are currently treated with small molecule tyrosine kinase inhibitors (TKIs) in the first line due to their exceptional response rate (70-80% ORR) and low toxicities⁸. These include tumors that contain EGFR, ALK, ROS, RET, MET, RAF and NTRK mutants, most of which are LADC ^9–12. Many tumors bearing these particular driver mutations are immunologically suppressive and respond poorly to immune checkpoint inhibitors (ICIs)^13,14. Unfortunately, these immunologically cold tumors also often progress within 3 months of treatment with TKI¹⁵, and TKI used together (or sequentially) have been shown to induce high rate of pneumonitis^16,17. Alternatively, tumors harboring no actionable driver are treated with single agent ICI if tumor expresses PD-L1 in 50% or more cells^18,19 whereas tumors expressing less than 50% of PD-L1 are treated with combination chemotherapy and immunotherapy. Patients who have failed targeted therapy are treated with chemotherapy, whereas those who have failed immunotherapy and chemotherapy are often considered for clinical trials. However, salvage docetaxel with or without ramucirumab, which is an antibody blocking vascular endothelial growth factor receptor 2 (VEGFR-2) protein, are often given when clinical trials are not available. The approval of ICI (e.g. anti-CTLA-4, anti-PD-1, anti-PD-L1…etc.) for metastatic melanoma in 2011²⁰, then later for NSCLC in 2016²¹, dramatically improved patient outcomes^22,23. However, the continued low rate of durable response to therapies in NSCLC underscores the major unmet need for improved treatment options. Moreover, it has been shown that patients with certain mutations (e.g. EGFR, KRAS, ALK, STK11) can have even worse rates of response for reasons still unknown^24–26. Investigation revealed that these tumors have low CD8+ T cells and low PD-1/PD-L1 expression ^24,27. While many concepts are being tested such as combination ICIs, these strategies may prove futile since NSCLC can be immunologically cold in certain cases²⁸. Further, TKI and ICI combination therapy has shown a high rate of severe side effects¹⁶.

The novel immunotherapy techniques of ACT have gained momentum over the last decade and continue to show promise moving forward. The three main types of ACT include chimeric antigen receptor T cell (CAR-T), modified T cell receptor (TCR), and TIL therapies (Figure 1). Both CAR and TCR therapies can pull T cells from peripheral blood, however, TIL therapy requires solid tumor infiltrates. Counter to standard therapeutics, ACT is a “living” treatment where re-infused lymphocytes can expand more than 1000-fold after administration and can remain in circulation for several years (or more)⁶. TIL as second line therapy (or above) in advanced stage cancers after failure of PD1/L1 checkpoint inhibitors appears to be feasible and effective^29,30. In melanoma, patients previously treated with BRAF/MEK inhibitors who had progression on these therapies were also included in the initial trial, and neither BRAF status nor PD-L1 status correlated with response to TIL therapy ³⁰. Therefore, combination use TIL therapy after progression on TKI may be an avenue of therapy to overcome TKI resistance, and offer longer term disease control. However, for NSCLC, tumor genotypes and phenotypes that will respond best to TIL therapy, and situations where TIL therapy and ICI therapy together will be effective, are currently unknown. Numerous trials combining ICIs and TIL therapy are underway, and will be discussed more in subsequent sections.

Figure 1: Adoptive cell therapy subtypes.

(A) CAR-T cells are now up to 5 generations, all containing CD3ζ chains and mixtures of co-stimulatory molecules (CM) or transgenes (TG). (B) Modified-TCR cells include altered variable alpha and beta chains of the TCR to recognize specific tumor (neo)antigens. (C) TILs are isolated from solid tumors, are polyclonal (indicated here by various colored TCRs), and can be cultured in bulk, further selected for specificity, or cultured for a shorter duration (young).

CAR-T Therapy

CARs (Figure 1A) are recombinant receptors that have been synthetically engineered into T cells, usually via retroviral infection, and provide targeted antigen binding as well as T cell activation^22,31. A similar process can also be completed with NK cells (CAR-NK therapy)³². To date, there are five generations (Gen) of CARs: 1^st Gen contain only a CD3ζ chain in their signaling domain with no co-stimulatory molecules (CM); 2^nd Gen have CD3ζ and one CM, allowing dual signaling pathways; 3^rd Gen have CD3ζ and several CMs; 4^th Gen resemble 2^nd Gen but incorporate an additional NFAT-responsive cassette that expresses anti-cancer cytokines (i.e. . triple signals - primary CD3ζ, CM, and expressed transgenic (TG) proteins); lastly, 5^th Gen are similar to 2^nd and 4^th Gen but add membrane receptors IL-2Rβ that provide a binding site for STAT3 and activate the JAK-STAT signaling domain³³. The CMs used often include CD28, CD27, CD134 (OX40), and CD137 (4-1BB)³⁴. While CAR-T therapies have shown significant durability in hematologic cancers such as B cell lymphomas, acute lymphocytic leukemia, and multiple myeloma^35–37, they have not been successful in solid tumors because of their requirement of antigen specificity²².

Modified TCR Therapy

The TCR is the antigen recognition structure that is expressed by all T cells and connects with antigen presenting cells (APCs) via the major histocompatibility complex (MHC) molecules on their surface. Upon binding, the T cells either recognize the antigen presented as self or foreign, and then they proceed accordingly³⁴. Unlike CARs, TCRs are naturally occurring and are somatically rearranged through V/D/J recombination to confer recognition of epitopes derived from proteins residing within any subcellular compartment^38,39. This enables TCRs to detect a broad spectrum of targets and, since TCRs have evolved to efficiently detect and amplify antigenic signals, these receptors are much more sensitive to smaller epitope densities than those required for CAR-signaling³⁴. With modified TCR therapy, a retrovirus is often used to transduce modified alpha and beta chains of the TCR to recognize tumor specific antigen(s) (Figure 1B)^32,34. Similar to CAR-T therapy, TCR therapy is restricted by its narrow scope of antigen recognition but has shown good results in melanoma, nasopharyngeal cancer, and cervical carcinoma⁴⁰.

TIL Therapy

The origins of clinical TIL research are credited to Dr. Steven A. Rosenberg beginning in the late 1980’s at the National Cancer Institute in Bethesda, Maryland^41–44. While the infiltrating nature of leukocytes was observed as early as the 1860’s^32,45, Rosenberg first isolated polyclonal TILs (Figure 1C) from mice in the early 1980’s and showed that pairing TILs with IL-2 could improve colon cancer in mice⁴⁶. High dose IL-2 (720,000 IU/kg) would go on to be approved for renal cell carcinoma and melanoma in 1992⁴⁷, but the progress for TILs was much slower due to the complex nature of the process, as well as skepticism of patient efficacy and tolerance. It was not until February 2024 that the first FDA approved TIL therapy⁴⁸, called lifileucel, to be used as second-line therapy in advanced melanoma. Cost remains a major area for improvement, because the newly approved treatment is estimated at over $500,000 USD per patient – more than some CAR-T treatments. A possible solution to this problem may be observed in our European counterparts, where TILs are produced in-house directly at the hospitals rather than outsourcing to companies^49,50 – an estimated cost of only $55,000 USD⁴⁸. However, regardless of the many challenges still ahead, this new groundbreaking approval of lifileucel will now pave the way for advances in other cancer types, like NSCLC. According to clinicaltrials.gov, there are already TIL trials involving cancers of the brain, liver, cervix, breast, pancreas, gastrointestine, colorectal, esophagus, and more.

Success of TIL therapy is dependent on numerous variables and mechanisms. First, perhaps most obvious, there must be tumor-reactive TIL present in the patients surgical or biopsy specimen. For this reason, it is often recommended that tumors must be greater than 1.5cm in diameter to ensure sufficient TIL counts for expansion but smaller masses could possibly be utilized^51,52. T cells must also maintain memory status to avoid being terminally differentiated into effector type T cells during the expansion process⁵³. Further, upon re-infusion TILs must navigate from the blood to the site(s) of remaining tumor that was not previously removed. They must then exit through endothelial venules according to a three-step process involving rolling via L-selectin, activation via CCR7, and sticking via LFA-1^54,55. Lastly, once they have arrived at their destination, they must selectively identify their target and induce effector functions (releasing perforin, granzyme B, IFN-γ, TNF-α,…etc.) to eliminate the cancer³⁹.

2. TIL Expansion and Infusion Protocols

At a surface level, the process of producing TILs appears straightforward. First, a patient’s tumor is resected and dissociated, then TILs are then expanded in culture with IL-2, tested for quality and reactivity, and finally re-infused into the patient (Figure 2). Yet, at each step there are multiple options for procedural variation, manual labor can be time consuming, and the space required to achieve the necessary TIL counts under good manufacturing practices (GMP) can quickly overcome a standard research lab. Additionally, there are various subsets of TILs, which depending on expansion, can accelerate or further complicate the process.

Figure 2: Schematic of simplified TIL process. Here we show a simplified overview of the TIL process in NSCLC.

Tumors are resected and dissociated, TILs are then expanded in culture with IL-2, tested for quality and reactivity, and finally re-infused into the patient. However, depending on the protocol chosen (bulk, selected, young, or hybrid), there are various caveats along the way that add to the complexity of development.

The initial steps are approximately the same for all TIL subtypes prior to the rapid expansion protocol (pre-REP). First, TILs can be acquired from enzymatic tumor digests OR tumor fragments. Next, cell cultures can be initiated in 24-well plates with 1x10⁶ tumor digest cells per well, one tumor fragment in 2 mL of complete medium (RPMI 1640 with glutamine, supplemented with 10% human AB serum, 25 mM HEPES buffer and 10 μg/mL gentamicin) and 6000 IU/mL of IL-2, OR using gas-permeable flasks⁵⁶. Then cells are incubated at 37°C in 5% CO₂. It typically takes ~1 week for the initial TIL cultures to overtake the tumor cells⁵⁷.

Bulk TILs vs. Selected TILs vs. Young TILs

At this point, a decision will need to be made regarding expansion in bulk, selected, or young TILs. Each type has unique advantages and disadvantages. In general, bulk TILs offer simplicity and broad responsiveness, selected TILs are more specific, and young TILs have more proliferative potential and persistence (Figure 3). Further, to overcome the possibility of T cells becoming terminally differentiated, it is possible to reprogram cells to a naïve state by: (1) reprogramming into induced pluripotent stem cells, then to naïve T cells, or (2) direct reprogramming from differentiated to less differentiated^53,58. Researchers are also exploring hybrid options of each technique²³. In general, the final result will primarily consist of tumor antigen reactive polyclonal CD8+ and CD4+ T cells^22,32.

Figure 3: TIL subtypes.

Depending on which rapid expansion procedure (REP) chosen, different TIL products can be achieved. Bulk TILs offer simplicity and broad responsiveness but can become exhausted or terminally differentiated (darker depiction). Selected TILs are more specific but can take longer because of a smaller starting population. Young (minimal) TILs have more proliferative potential and persistence (lighter depiction) but have fewer cell totals at infusion.

Bulk TILs are expanded in totality, regardless of which subset of lymphocyte they are associated with. The heterogeneous population is composed of CD8+, CD4+ (Th (T helper)1, Th2, Th9, Th17, Th22, Treg (regulatory T cells), and Tfh (follicular helper T cells)), natural killer (NK) cells, myeloid-derived suppressor cells (MDSCs), tumor-associated macrophages, B cells, and other elements of the tumor environment⁵⁹. Importantly, the culturing conditions provide an environment most favorable to T cells, so while other cell types may be present at the final infusions step, they are at very low levels. Bulk would also comprise naïve, activated, and exhausted cells, which leads to bulk TILs requiring large numbers of cells (5 – 500 x 10⁹ cells or more) for infusion to achieve best results. In total, the process for bulk TIL therapy could take 4-6 weeks start-to-finish, but new industrial advancements are improving long wait times^29,52,57.

Selected TILs consist of antigen-specific TILs or can be sorted according to marker specific collections. These can include CD8+, CD4+, CD28+, CD27+, CD134+ (OX40), CD137+ (4-1BB), CD279+ (PD-1), specific neoantigens (e.g. mutations in TP53, KRAS, …etc.)^60–62, as well as negative selections (e.g. exclude FOXP3+) and many more^59,60,63,64. Following the pre-REP phase, TILs will be tested for reactivity against specific (neo)antigens using ELISPOT, flow cytometry, or tetramer staining⁵⁹. Similarly, they can be sorted according to surface markers using flow cytometry or magnetic sorting. These filtered sets then undergo REP, but starting at a much smaller total population, which leads to longer end-to-end preparation time to achieve similar cell totals as bulk at infusion⁶¹.

Lastly, young TILs are less differentiated and have shorter expansion times compared to bulk and selected TILs^50,65,66. This allows for a higher proliferative capacity and longer persistence. Young TILs are typically defined by cultures that had only expanded to confluency of the original 2-mL well and had eliminated their co-cultured tumor cells⁶⁶. Therefore, pooling all of the wells in a single 24 well plate could achieve approximately 5 x 10⁷ young TIL cells. These cultures usually take 10-18 days after initiation, compared to the multi-week process for bulk and selected TILs. Notably, the alternative long culture times required to perform screening assays showed telomere shortening as well as reduced expression of CD27 and CD28 in the TIL cultures^50,65,66.

Rapid Expansion Protocol (REP)

There are now multiple solutions to the complicated TIL process, including automated biotech solutions (e.g. Miltenyi Biotec) and better basic lab tools (e.g. Wilson Wolf⁵⁶). Rosenberg and Jin et al. detailed the two common options for the REP process⁵⁷. Traditionally, in T-175 flasks there are 1×10⁶ TILs suspended in 150 mL of complete media. The TILs are then cultured with irradiated allogeneic peripheral blood mononuclear cells (PBMC) as “feeder” cells. Next, the cells are cultured in a 1:1 mixture of complete medium and AIM-V medium (50/50 medium), supplemented with 3000 IU/mL of IL-2 and 30 ng/mL of anti-CD3. The T-175 flasks are incubated at 37°C in 5% CO₂, and half of the media is changed on day 5 using 50/50 medium with 3000 IU/mL of IL-2. On day 7, TILs from two T-175 flasks are combined in a 3liter bag in 300 mL of AIM V media supplemented with 5% human AB serum and 3000 IU per mL of IL-2. Every day, TILs are re-counted and a corresponding amount of fresh media is addedso that TIL concentration remains between 0.5 and 2.0×10⁶ cells per mL.

Alternatively, a “simplified” method utilizes gas-permeable flasks⁵⁷. A major limitation of the traditional method is flask height, which restricts how many TILs can expand in one flask. To combat this problem, one can use gas-permeable flasks that are vertically taller and cylindrical. In 500 mL capacity flasks , 5-10×10⁶ TILs are cultured with irradiated allogeneic PBMC in 400 mL of 50/50 medium, supplemented with 5% human AB serum, 3000 IU/mL of IL-2 and 30 ng/mL of anti-CD3, then incubated. On day 5, 250 mL of supernatant is removed and centrifuged. The TIL pellets are re-suspended with 150 mL of fresh medium with 5% human AB serum, 3000 IU/mL of IL-2, and added back to the original flasks. Then, TILs are expanded serially on day 7 and supplemented with fresh media after 4 days. Finally, the cells are harvested on day 14 of culture.

Post-REP Infusion

After TILs have undergone successful REP, they are ready for infusion back into the patient. Prior to infusion, there is typically a round of lymphodepleting chemotherapy 2-7 days beforehand. This pretreatment includes cyclophosphamide and fludarabine (cy/flu) or total body irradiation (TBI) at levels that are non-myeloablative^52,67. It has been shown that treatment with cy/flu increases efficacy of the TIL treatment, because the suppressing impact of regulatory cells is dampened by increasing host homeostatic cytokines (e.g. IL-7 and IL-15) as well as decreasing endogenous lymphocyte and myeloid populations^41,51,59. Once the TILs are infused, patients undergo high doses of IL-2 (720,000 IU/kg) to help boost TILs. To manage side-effects of IL-2, protocols often elect to administer less than 6 high doses post infusion or use low/intermediate doses over longer periods^29,59.

Epigenetic control of T cell Expansion and Exhaustion

As discussed above, one of the challenges of TIL therapy is to expand effector T cells that will remain functional, while limiting regulatory T cells and T cell exhaustion. All these phenotypes are controlled in part through epigenetic mechanisms, including the methylation of DNA and histones to silence gene expression. For example, multiple studies have shown that trimethylation of histone 3 at lysine 27 (H3K27me3), which is catalyzed by the histone methyltransferase EZH2, favors Tregs in the tumor microenvironment ^68,69. In addition, H3K27me3 can suppress transcription of genes associated with memory T cells, including the transcription factors Tcf7 and Id3 ^53,70. Specifically relevant to TILs, targeting EZH2 with pharmacological inhibitors may help improve the efficacy of the therapy by preventing terminally differentiated and exhausted phenotypes that result from extensive ex vivo expansion ^71,72. Similarly, use of ex vivo BET bromodomain inhibitors have been shown to enhance stem like and central memory phenotypes, downregulate factors associated with Th17 differentiation, and allow for improved persistence and anti-tumor activity of re-infused T cells ⁷³. Additionally, inhibition of EZH2 ⁷⁴, as well as other epigenetic targets such as DNA methyltransferases (DNMTi) ⁷⁵, or histone deacetylases (HDACi) ⁷⁶, can improve patient response to immune checkpoint inhibitors, and therefore have potential to help improve outcomes of TIL infusions, particularly when combined with immune checkpoint inhibitors ⁵³. Given that deletion of Ezh2 can also restrict memory T cell function, finding the appropriate doses and timing of drugs will be critical ^70,77. Although more research is needed to validate targeting epigenetic programs in TIL protocols, data from many groups suggest that epi-drugs will have clinical usefulness.

3. Clinical success and current trials in nsclc

Initial trials (primarily in melanoma) in the 1980s yielded disappointing outcomes. Some trials reported that none of the patients achieved greater than 50% reduction of tumor burden⁷⁸, and others showed similar results because of inability to expand TILs or non-optimized protocols^22,79,80. However, after more initial demonstrations of hopeful outcomes, multiple trials around the globe began confirming results with objective response responses (ORR) consistently between 40-70% ⁸¹. Rosenberg and colleagues were able to show in several consecutive trials that ORRs of up to 72% were achievable, 10-20% of patients had complete remission, and 40% had durable clinical responses^81–83. Importantly, the probability of these responses did not appear to depend to previously failed systemic treatment.

As previously mentioned, lifileucel (LN-145 in Table 1) was the first TIL therapy to earn FDA approval in February 2024 for advanced melanoma based on results from the C-144-01 study²⁹. According to a follow-up analysis ^84,85, among all patients (n=153), the median overall survival (OS) was 13.9 months (95% CI, 10.6-17.8), and the 4-year OS rate was 22.2%. Within the responders (n=48), 4-year OS rates by response type were: 48.3% in early responders (95% CI, 31.9%-62.9%, n=39), 41.7% in late responders (95% CI, 10.9%-70.8%, n=9), 37.2% in responders without a deepened response (95% CI, 21.0%-53.5%, n=16), and 68.2% in responders with a deepened response (95% CI, 39.5%-85.4%, n=32). Early and late response were defined as before and after 42 days, respectively, and a deepened response was those patients who had an increased in response over time. The median duration of response (DOR) for all responders was not reached (NR; 95% CI, 8.3-NR). Specifically, median DOR according to response type were: NR in early responders (95% CI, 6.1-NR), 19.8 months (95% CI, 4.1-NR) in late responders, 26.2 months (95% CI, 4.1-NR) in responders without a deepened response, and NR (95% CI, 8.3-NR) in responders with deepened responses ⁸⁴. All patients in this study had progressed on (or after) anti-PD-1/L1 therapy, and 49.7% of patients had tumors with greater than 1% PD-L1+ tumor proportion score.

Table 1: Search of NSCLC TIL trials in the US.

The following table includes a summary of clinical trials with TIL therapy according to clinicaltrials.gov as of July 2024.

NCT Number	Study Title	Brief Summary From ClinicalTrials.Gov	Interventions
NCT03215810 Phase 1 Completed	Nivolumab and Tumor Infiltrating Lymphocytes (TIL) in Advanced Non-Small Cell Lung Cancer	“Investigators plan to study the safety, side effects, and benefits of tumor-infiltrating lymphocytes (TILs) when they are given with the drug nivolumab. Nivolumab is a type of immunotherapy - a drug that is used to boost the ability of the immune system to fight cancer, infection, and other diseases.”	TIL / Nivolumab
NCT02848872 Observational Completed	Evaluation of Tumor and Blood Immune Biomarkers in Resected Non-small Cell Lung Cancer	“The hypothesis of this study is that functional tumor infiltrating lymphocyte (TIL) isolation from resected lung cancer specimens is feasible, allowing determination of tumor antigen-specific T cell reactivities. The primary objective of this study is to investigate the feasibility of isolating functional tumor infiltrating lymphocytes s(TILs) to determine tumor antigen-specific T cell re-activities in 30 resected lung tumor specimens. Successful isolation of TILs will be defined as collecting 1x10−6 viable, CD45+ mononuclear cells or greater from tumors containing \>/=1 gram of excess tissue. If successful isolation of TILs can be obtained from \>/= 66% of resected tumor specimens, the protocol will be considered feasible. The primary exploratory objective is to identify immunologic signatures that predict clinical outcomes from cytotoxic chemotherapy and/or immunotherapy.”	TIL
NCT01820754 Phase 2 Completed	Evaluation of Circulating T Cells and Tumor Infiltrating Lymphocytes (TILs) During / After Pre-Surgery Chemotherapy in Non-Small Cell Lung Cancer (NSCLC)	“The purpose of this study is to evaluate whether the combination of neoadjuvant chemotherapy (chemotherapy before surgery) plus ipilimumab for lung cancer increases the number of patients with detectable circulating T cells with specificities against tumor associated antigens (TAA) from zero percent (0%) of patients prior to therapy to 20% of patients after neoadjuvant chemotherapy plus ipilimumab.”	TILs / Ipilimumab / Paclitaxel / Cisplatin / Carboplatin
NCT05397093 Phase 1 Active - not recruiting	ITIL-306 in Advanced Solid Tumors	“ITIL-306-201 is a multicenter, clinical trial evaluating the safety and feasibility of ITIL-306 in adult participants with advanced solid tumors whose disease has progressed after standard therapy. ITIL-306 is a cell therapy derived from a participant’s own tumor-infiltrating immune cells (lymphocytes; TILs) and contains a unique molecule designed to increase TIL activity when it encounters folate receptor α (FOLR1) on the tumor.”	ITIL-306
NCT02818920 Phase 2 Active - not recruiting	Neoadjuvant Pembrolizumab	“This multi-institutional clinical trial is studying two doses of pembrolizumab administered prior to surgery (neoadjuvant therapy) and 4 doses administered after surgery (adjuvant therapy) for stage IB, II or IIIA non-small cell lung cancer. Pembrolizumab is a type of immunotherapy that may enhance the ability of the immune system to fight off cancer. The study will investigate the effects of pembrolizumab on the immune system and how certain immune cells, called TILs (tumor infiltrating lymphocytes), respond to pembrolizumab. Previous studies suggest that pembrolizumab could alter the immune cells in a way that the the immune cells identify cancer cells. Pembrolizumab has been approved for the treatment of advanced lung cancer, but is investigational in this setting.”	TIL / Pembrolizumab
NCT05681780 Phase 1/2 Recruiting	Clinical Trial of CD40L-Augmented TIL for Patients With EGFR, ALK, ROS1 or HER2-Driven NSCLC	“To determine the effect of a special preparation of cells, called tumor-infiltrating lymphocytes (TIL) stimulated with CD40L, when given with the drug nivolumab, for patients with EGFR, ALK, ROS1, or HER2-genomically altered lung cancer.”	TIL / Nivolumab
NCT04614103 Phase 2 Recruiting	Autologous LN-145 in Patients With Metastatic Non-Small-Cell Lung Cancer	“This is a prospective, open-label, multi-cohort, non-randomized, multicenter study evaluating LN-145 in patients with metastatic non-small-cell lung cancer.”	LN-145 (TIL)
NCT05573035 Phase 1 Recruiting	A Study to Investigate LYL845 in Adults With Solid Tumors	“This is an open-label, multi-center, dose-escalation study with expansion cohorts, designed to evaluate the safety and anti-tumor activity of LYL845, an epigenetically reprogrammed tumor infiltrating lymphocyte (TIL) therapy, in participants with relapsed or refractory (R/R) metastatic or locally advanced melanoma, non-small cell lung cancer (NSCLC), and colorectal cancer (CRC).”	LYL845 (TIL)
NCT05576077 Phase 1 Recruiting	A Study of TBio-4101 (TIL) and Pembrolizumab in Patients With Advanced Solid Tumors	“A multicenter trial to investigate TBio-4101, an autologous, neoantigen-selected, tumor-reactive TIL product, in patients with advanced solid malignancies.”	TBio-4101 (TIL) / Pembrolizumab
NCT03645928 Phase 2 Recruiting	Study of Autologous Tumor Infiltrating Lymphocytes in Patients With Solid Tumors	“A prospective, open-label, multi-cohort, non-randomized, multicenter study evaluating adoptive cell therapy (ACT) with TIL LN-144 (Lifileucel)/LN-145 in combination with checkpoint inhibitors or TIL LN-144 (Lifileucel)/LN-145/LN-145-S1 as a single agent therapy.”	LN-145 (TIL) / Pembrolizumab / Ipilimumab / Nivolumab
NCT02133196 Phase 2 Recruiting	T Cell Receptor Immunotherapy for Patients With Metastatic Non-Small Cell Lung Cancer	“The purpose of this study is to see if these specifically selected tumor fighting cells can cause non-small cell lung cancer (NSCLC) tumors to shrink and to see if this treatment is safe.”	Young TIL
NCT06060613 Phase 1/2 Recruiting	Safety and Efficacy of OBX-115 in Advanced Solid Tumors	“This is a study to investigate the safety and efficacy of an investigational OBX-115 regimen in adult participants with advanced solid tumors.”	OBX-115 (TIL)
NCT05361174 Phase 1/2 Recruiting	A Study to Investigate the Efficacy and Safety of an Infusion of IOV-4001 in Adult Participants With Unresectable or Metastatic Melanoma or Stage III or IV Non-small-cell Lung Cancer	“This is a study to investigate the efficacy and safety of an infusion of IOV-4001 in adult participants with unresectable or metastatic melanoma or advanced non-small-cell lung cancer (NSCLC).”	IOV-4001 (TIL)
NCT05566223 Phase 1/2 Not yet recruiting	CISH Inactivated TILs in the Treatment of NSCLC	“A clinical trial to assess the safety and efficacy of genetically-engineered Tumor Infiltrating Lymphocytes (TIL) in which the intracellular immune checkpoint CISH has been inhibited using CRISPR gene editing for the treatment of Metastatic Non-small Cell Lung Cancer (NSCLC).”	CISH Inactivated TIL / Pembrolizumab

Lifileucel is currently being studied for efficacy and safety in patients with metastatic NSCLC patients who are resistant to ICIs (PD-L1–negative, low tumor mutational burden, STK11 mutation,… etc.) and are treatment refractory (NCT03645928). At phase 2 of this multicenter study, the objective response rate was 21.4%, which is a major improvement for this population of patients⁸⁶. Other recent studies have demonstrated growing improvement in the strategy and feasibility within NSCLC patients – including those with refractory disease^30,87. As of July 2024, there have been (or currently are) 14 US clinical trials related to TIL therapy in NSCLC with a variety of combination studies and genetically altered TILs since 2000 (clincialtrials.gov). Of these trials, 3 have been completed – 2 of which only analyzed properties of TIL rather than used TIL as a treatment mechanism. In Table 1, we summarize each trial with its respective treatment – many include combination treatments with ICIs or TKIs.

The lone completed trial utilizing TILs as therapy (NCT03215810) was conducted as a single-arm open-label phase 1 trial of TILs administered with nivolumab in 20 patients with advanced NSCLC following initial progression on nivolumab monotherapy³⁰. Their primary endpoint was safety (achieved with severe toxicity rate ≤17% (95% confidence interval, 3–29%)) and secondary end points were ORR, DOR and T cell persistence. Among the 13 patients who could be evaluated, 3 showed confirmed responses, 11 experienced a reduction in tumor size (−35% median best change), and 2 patients achieved complete responses (sustained 1.5 years later). Further analysis showed that T cells targeting various cancer mutations were identified after TIL treatment and were more prevalent in patients who responded to the therapy. Thus, Creelan et al. showed that TILs are generally safe, show clinical activity, and are a promising new treatment option for NSCLC³⁰.

Challenges for toxicity and patient efficacy

While the prospect of TIL therapy in NSCLC is very bright, there still remain drawbacks and challenges. Advances in technology have shown drastic improvement the REP process, as well as identifying the subcategories of selected TILs for optimal reactivity, plasticity, stemness, and resilience^{57,64,66,88,89}. With respect to production, average turnaround is 4-6 weeks (possibly longer). Such long wait times may not be feasible for patients with extreme late-stage disease, as seen with the loss of two patients in the Creelan et al. study, where TIL production was approximately 63 days^30,90. Furthermore, those with heavy tumor burden, fast progression, and advanced age may find the treatment regimen with IL-2 and cy/flu very hard to tolerate even over a short period, with four patient deaths occurring on the C-144-01 study attributed to these treatments²⁹. The study of 16 TIL treated NSCLC patients in the Creelan et al. study, found main side-effects to be hypoalbuminemia (13 - grade 2; 1 - grade 3), hypophosphatemia (4 - grade 2; 8 - grade 3), nausea (7 - grade 1; 7 - grade 3), hyponatremia (8 - grade 1; 2 - grade 3; 1 - grade 4), and diarrhea (5 - grade 1; 4 - grade 2)³⁰. Interestingly, cytokine release syndrome appeared as a rare side-effect. Again, the majority of TIL-related adverse events were related to lymphodepletion and IL-2, but peaked around 7 days post-infusion and resolved within one month. Notably, the recruitment for the study included: 50% current/former smoker, median age 54 years, median PD-L1 proportion 6%, many patients with targetable mutations, predominant LADC histology, median nonsynonymous TMB of 1.5 mutations per megabase, and mostly bulky disease. Thus, a broader cohort for better analysis is still needed.

A common course of TIL treatment includes cyclophosphamide for two days, then fludarabine for 5 days, followed by TIL infusion and IL-2⁶. Pretreatment with cy/flu will deplete the immune system to reduce endogenous impact on TILs (resulting in leukopenia, lymphopenia, neutropenia…etc.), but the immune system is reconstituted within just over one week^6,30. As the field continues to develop, substantial hurdles remain to be crossed with regard to both the TIL expansion and reinfusion process. Researchers are aiming to achieve the most effective TILs, while also minimizing adverse impacts of pre- and co-treatment therapeutic regimens with cy/flu and IL-2. For example, IL-2 can lead to terminal effector state of T cells and, to circumvent the production of predominantly exhausted T cells, some have suggested using homeostatic cytokines like IL-7, IL-15, or IL-21⁸¹. Further, it has been shown that an elevated number of IL-2 doses (720,000 IU/kg every 8 hours up to 15 doses) can actually increase Treg counts and increase risk of cytokine release syndrome⁹¹.

Concluding remarks

As lung cancer continues to harm hundreds of thousands of patients annually, it remains of utmost importance to find personalized treatments for patients that are curated to their specific tumor microenvironment. Arguably the most patient specific approach currently in development is using a patient’s endogenous TILs, utilizing their own natural mechanisms to fight the cancer. Better protocols, modern technology, epigenetic reprogramming, and improved administration are on the rise, all of which paint a highly optimistic future for better treatment of the world’s most prevalent and deadly cancer.

Highlights.

• Tumor-infiltrating lymphocytes (TILs) are a promising precision therapy for lung cancer

• TIL subtypes and age can be influenced by expansion protocols

• Inhibitors of epigenetic enzymes could improve TIL expansion and therapeutic effect

• Numerous trials are ongoing for TILs in lung cancer