Reduced dose fludarabine and cyclophosphamide lymphodepletion before tumor-infiltrating lymphocyte therapy in melanoma

ABSTRACT

Metastatic melanoma patients who have progressed following targeted or immunotherapy have a dismal prognosis; however, novel tumor-infiltrating lymphocytes (TIL) therapy (lifileucel) with traditional lymphodepletion (LD) has demonstrated durable responses and improved survival. We hypothesize that a reduced dose of fludarabine (Flu)/cyclophosphamide (Cy) LD before TIL therapy will have similar TIL expansion and persistence post-infusion with a reduced toxicity profile. This study is a single-center, open-label phase II pilot trial evaluating the in vivo persistence, efficacy, and safety of TIL therapy after reduced-dose Flu/Cy LD. Key inclusion criteria include unresectable or metastatic melanoma with disease progression after one or more lines of therapy. Patients will receive Flu (30 mg/m² on days  −4,  −3,  −2,  −1) and Cy (750 mg/m² on days  −4,  −3,  −2) LD and TIL infusion (day 0) followed by high-dose IL-2 (600,000 IU/kg for six doses). The primary objective will be to ascertain TIL persistence using T-cell receptor sequencing at day 42. The secondary objectives will include the efficacy parameters, including response rate, duration of response, progression-free survival, and overall survival, as well as the safety profile of TIL with a reduced dose of Flu/Cy LD regimen. Exploratory objectives will involve correlative analyses to characterize the microenvironment.

Clinical Trial Registration

NCT06151847.

1. Introduction

1.1. Tumor-infiltrating lymphocyte therapy

Adoptive cell immunotherapy using tumor-infiltrating lymphocytes (TIL) consists of isolation of TIL from fresh tumor sample, interleukin (IL)-2 mediated selection of activated cells expressing IL-2 receptors, and ex-vivo expansion with subsequent administration of the expanded TIL product to the patient. TIL infusion is preceded by non-myeloablative lymphodepletion (NMA-LD) chemotherapy, consisting of cyclophosphamide (Cy) 60 mg/kg for 2 days cyclophosphamide and fludarabine (Flu) 25 mg/m² for 5 days, to reduce the number of regulatory T cells, creating a hostile tumor microenvironment, followed by adjuvant high-dose IL-2 to assist with in vivo engraftment. As with other immunotherapy approaches, TIL therapy has been most extensively utilized in melanoma, where response rates of approximately 50% have previously been reported [1–4]. In the setting of TIL therapy after prior immune checkpoint inhibitor (ICI), observed response rates are lower, possibly due to effects of the prior therapy or patient selection [5]. Response rates from melanoma patients previously treated with ICI therapy are in the 30–35% range [6–8]. Nonetheless, TIL therapy has been shown to be an effective salvage regimen and can be effective in patients with ICI-refractory disease.

Widespread use of TIL therapy has been limited by several factors, including the expense and regulatory expertise required for Good Manufacturing Practice (GMP)-certified facilities for cell growth, the need for high-dose IL-2 that may result in severe toxicities such as capillary leak syndrome with potential for multi-organ failure and need for expertise and intensive care [9,10], and NMA-LD with the risk for cytopenia and poor immune reconstitution which may lead to requirement for infection precautions, transfusions and growth factor support, and may cause life-threatening complications [2,3].

Centralized TIL manufacturing with cryopreservation has the potential to offer TIL therapy to a broader population of patients and to optimize cell growth procedures. In a Phase II study using centrally manufactured TIL in 153 metastatic melanoma patients sponsored by Iovance Biotherapeutics, the cell product lifileucel produced an overall response rate (ORR) of 31% and median duration of response was not reached after 27.6 months follow-up, with 42% of responses maintained at or after 18 months [11]. All these patients had prior ICI therapy and were refractory to a median of three lines of prior therapies. In the updated report, including 153 patients with 4-year follow-up, an ORR of 31% (n = 48) was noted, the median duration of response (DOR) was not reached, and 4-year overall survival (OS) was 22% [12]. The side effects of this regimen are due to the known adverse event profile of lymphodepletion and high-dose-IL-2 [8]. These compelling data demonstrate successful treatment of a patient population with high unmet needs and confirm that centralized TIL manufacturing is feasible and effective. In the trial mentioned above, grade 3 or 4 adverse events due to the NMA-LD were common. Specifically, grade 3 or 4 thrombocytopenia, anemia, and febrile neutropenia occurred in 82%, 56%, and 55% of patients, respectively. Additionally, due to the high-dose Cy and need for intravenous (IV) fluids and mesna administration for bladder chemoprotection, this regimen is administered inpatiently, leading to a significant increase in the length and expense of the hospital stay.

1.2. Reduced dose lymphodepletion

While there is ample evidence to suggest the biological need for lymphodepleting chemotherapy to help eliminate cytokine sinks and reduction in the number of circulating regulatory T cells and myeloid-derived suppressor cells and to promote expansion, function, and persistence of adoptively transferred T cells, few studies have focused on modifying the traditional doses of NMA-LD chemotherapy for TIL [1–3,13]. One study, conducted at Sheba Medical Center, trialed three different conditioning regimens for metastatic melanoma patients including the traditional Cy 60 mg/kg × 2 days and Flu 25 mg/m² × 5 days (120 Cy/125 Flu), Cy 30 mg/kg × 2 days and Flu 25 mg/m² × 5 days (60 Cy/125 Flu), and total body irradiation (TBI) with Flu 25 mg/m² × 3 days (TBI/75 Flu). The authors concluded that bone marrow depletion was most profound in the patients who received either 120 Cy/125 Flu or 60 Cy/125 Flu; however, recovery was quicker; thus, toxicity and duration in the hospital were lower with 60 Cy/125 Flu-treated patients. Although ORR to TIL on 120 Cy/125 Flu was 28% and no responses were seen in the 60 Cy/125 Flu cohort, this discrepancy in response was thought to be due to the more heavily pre-treated status of enrolled patients on the 60 Cy/125 Flu cohort [14]. Recently, chimeric antigen receptor (CAR) T-cell therapy has been utilized successfully in various hematologic malignancies. Based on compelling biologic data, a reduced dose of FluCy lymphodepletion (LD) chemotherapy has been used in these patients. Specifically, studies showed an association between an increase in serum IL-15 levels post-lymphodepletion and clinical response after anti-CD19 CAR T-cell therapy [15,16]. In lymphoma patients, anti-CD 19 CAR T-cell therapy with axicabtagene ciloleucel and brexucabtagene autoleucel uses Cy 500 mg/m² and Flu 30 mg/m² for 3 days [17,18], lisocabtagene maraleucel uses Cy 300 mg/m² and Flu 30 mg/m² for 3 days [19], and tisagenlecleucel uses Cy 250 mg/m² and Flu 25 mg/m² for 3 days [20], as LD. In myeloma patients, anti-BCMA CAR T-cell therapy with idecabtagene vicleucel and ciltacabtagene autoleucel uses Cy 300 mg/m² and Flu 30 mg/m² for 3 days as LD [21,22]. A lower dose of Cy (<1000 mg/m²) does not require mesna, and LD regimens with Cy doses less than 1000 mg/m² can be safely administered as an outpatient.

As detailed above, several LD conditioning regimens have been used with TIL therapies. NMA-LD regimens have included cyclophosphamide/fludarabine, total body irradiation, or combining the two. The NMA-LD preconditioning regimen used in the lifileucel registration trial is based on the method developed and tested by the NCI, which involves 2 days of cyclophosphamide followed by 5 days of fludarabine [8]. Our proposed low-dose regimen is based on moderate intensity compared to current TIL regimens and those used before CAR T-cell therapies in hematologic malignancies. We selected a dose of intermediate-intensity lymphodepletion compared to current CAR T-cell therapy regimens and TIL therapy. We used the LD regimen dose based on biological rationale and prior experience with LD chemotherapies before TIL and CAR T-cell therapies; the current study will provide seminal data for future investigation. (Table 1)

Table 1.

Non-myeloablative lymphodepleting chemotherapy regimens.

		Cyclophosphamide			Fludarabine
Regimen	Reference	mg/kg	days	Total dose (mg)	mg/m2	days	Total dose (mg)
TIL LD Regimens
NCI High-dose LD TIL regimen	[14]	60	−7 to  −6 (2)	9540	25	−5 to  −1 (5)	237.5
NCI Low-dose LD TIL regimen	[14]	30	−5 to  −4 (2)	4770	25	−5 to  −1 (5)	237.5
Proposed TIL LD Regimen		mg/m2	days	Total dose (mg)	mg/m2	days	Total dose (mg)
RD-NMA-LD TIL Regimen		750	−4 to  −2 (3)	4275	30	−4 to  −1 (4)	228
CAR-T LD Regimens		mg/m2	days	Total dose (mg)	mg/m2	days	Total dose (mg)
CAR-T Regimen 1 (axicabtagene ciloleucel and brexucabtagene autoleucel)	[17,18]	500	−5 to  −3 (3)	2850	30	−5 to  −3 (3)	171
CAR-T Regimen 2 (lisocabtagene maraleucel, idecabtagene vicleucel and ciltacabtagene autoleucel)	[19,21,22]	300	−5 to  −3 (3)	1710	30	−5 to  −3 (3)	171
CAR-T Regimen 3 (tisagenlecleucel)	[20]	250	−5 to  −3 (3)	1425	30	−5 to  −3 (3)	171

Comparison is based on a person weighing 175 lbs, stands 5’10’ tall, converting to a person weighing 79.5 kg, and has a body surface area of 1.9 m². Cyclophosphamide difference between the current Iovance regimen and the proposed R-LD regimen is: 9540 mg  −  4275 mg = 5265 or (45% of the current dose). Fludarabine difference between current Iovance regimen and proposed RDL regimens 237.5 mg  −  228 mg = 9.5 mg or (96% of the current dose).

Abbreviations: TIL, tumor-infiltrating lymphocytes therapy; NCI, National Cancer Institute; CAR-T, chimeric antigen receptor T-cell therapy; LD, lymphodepletion; reduced dose non-myeloablative lymphodepletion (RD-NMA-LD).

This pilot trial thus proposes a reduced dose NMA-LD (RD-NMA-LD) with cyclophosphamide 750 mg/m² on days  −4,  −3,  −2 and fludarabine 30 mg/m² on days  −4,  −3,  −2,  −1 in melanoma patients receiving TIL therapy. One of the fundamental biological features of lymphodepletion is the ability to encourage long-term TIL persistence. The proportion of unique TCR clonotypes from the Lifileucel TIL therapy lots contributing to the peripheral blood T Cell Receptor (TCR) repertoire among infused patients was analyzed using a semi-quantitative polymerase chain reaction (PCR) followed by next-generation sequencing (NGS). The proportion of TCR clones composed of clonotypes identified in the product increased from a mean of 16% (n = 125) at pre-infusion to 83% at day 4 after TIL therapy infusion. The TCR clones declined to 51% at day 14 (n = 51) and remained 37% to 41% from day 42 (n = 120) to month 12 (n = 37) post-infusion of TIL therapy. No significant correlation was found between TIL persistence and efficacy (unpublished data, n = 153) [23]. We will also explore the impact on absolute lymphocyte count (ALC) with RD-NMA-LD, as ALC < 100/uL has been associated with adequate bone marrow suppression and lymphodepletion [14]. This trial will include detailed analyses of TIL persistence to characterize the reduced-dose regimen’s biological effects adequately.

1.3. Rationale

Using RD-NMA-LD for TIL therapy will make treatment safer and thus more applicable to a broader patient population, lessening the need for transfusion and growth factor support, and shortening the length and expense of hospitalization. As mentioned above, this pilot clinical trial will utilize the outpatient RD-NMA-LD regimen with centrally manufactured TIL infusion on day 0, followed by up to 6 doses of high-dose IL-2. The pilot exploratory trial intends to ascertain this regimen’s biological effect on TIL persistence using TCR sequencing technology. Detailed data on TIL persistence and lymphocyte subset analysis will provide critical insights into the biological effects of the reduced dose regimen. However, the small sample size currently limits our ability to effectively measure the response to therapy. A primary endpoint based on efficacy will be evaluated in a larger follow-up trial that will have adequate statistical power. We hypothesize that an RD-NMA-LD before lifileucel TIL therapy will have similar TIL expansion and persistence post-infusion compared to NMA-LD and may lead to similar efficacy with a reduced toxicity profile with RD-NMA-LD as compared to traditional NMA-LD.

1.4. Objectives

1.4.1. Primary objective

The primary objective will be determining the percentage of total TIL clones measured by the TCR population shared between the lifileucel TIL product and peripheral blood mononuclear cells (PBMCs) at day  +42 ( +7 days).

1.4.2. Secondary objectives

The secondary objectives include:

• To evaluate the efficacy parameters of lifileucel in combination with a reduced-dose lymphodepletion in patients with unresectable or metastatic melanoma by assessing ORR, DOR, progression-free survival (PFS), and OS.

• To characterize the safety profile of lifileucel in combination with a reduced dose lymphodepletion regimen in patients with unresectable or metastatic melanoma by assessing treatment-emergent adverse events (TEAE) and serious adverse events (SAE).

1.5. Trial design

This study is a phase II, open-label, single-center, single-arm pilot trial testing the in vivo persistence of lifileucel after RD-NMA-LD. Patients will undergo TIL procurement with a surgical excision of at least a 1.5 cm tumor specimen, which will be sent for centralized TIL manufacturing. The patient will receive reduced dose lymphodepletion consisting of Cy 750 mg/m² on days  −4,  −3,  −2 and Flu 30 mg/m² on days  −4,  −3,  −2,  −1 as an outpatient. Patients will receive inpatient TIL infusion on day 0, followed by high-dose IL-2 (600,000 IU/kg up to 6 doses). (Figure 1) Blood collection for TCR sequencing will occur to ascertain the persistence and diversity of T cell clones compared to lifileucel. On-treatment post-TIL infusion biopsies will also be obtained to ascertain the persistence and diversity of TIL in tumors compared to the procurement sample. The collected blood and tumor samples will help determine if the RD-NMA-LD is detrimental to the persistence and diversity of TIL therapy. Efficacy and safety outcomes will be assessed. Plasma will also be collected at scheduled time points for future correlative analyses.

Figure 1.

Schematic of trial design. Reduced dose lymphodepletion (outpatient): cyclophosphamide (cy) 750 mg/m² on days  −4, −3, −2 with Fludarabine (flu) 30 mg/m² on days  −4, −3, −2, −1. Tumor-Infiltrating Lymphocytes (TIL) administration with interleukin (IL)-2 (inpatient): TIL infusion (lifileucel) on day 0, followed by a short course of IL-2 (600,000 IU/kg every 8–12 hours for up to 6 doses days  +1 to  +3).

2. Methods

2.1. Study setting

The open-label, single-center, single-arm pilot trial will be conducted at the University of Kansas Cancer Center (KUCC) in Kansas City, KS.

2.2. Eligibility criteria

2.2.1. Inclusion criteria

Adult patients (age ≥18 years) with unresectable or metastatic melanoma (stage IIIC or IV) with disease progression after one to three lines of therapy including a programmed cell death protein-1 (PD-1) blocking antibody (and if BRAF V600 mutation positive, a BRAF inhibitor) with life expectancy of ≥3 months, ECOG Performance Status 0–1, at least one measurable target lesion, at least one resectable lesion ≥1.5 cm in diameter to generate TIL with minimal morbidity, adequate organ function defined as absolute neutrophils > 0.5 K/uL, platelets > 30 K/uL, hemoglobin ≥8 g/dL, serum alanine and aspartate transaminase (ALT/AST) ≤3 times the upper limit of normal (ULN) (≤5 times ULN in patients with liver metastasis), creatinine clearance (CrCl) ≥40 mL/min using the Cockcroft- Gault formula, and total bilirubin ≤2 mg/dL (≤3 mg/dL in patients with Gilbert’s syndrome), recovered from all prior therapy-related AEs to ≤grade 1, except for alopecia or vitiligo, washout period of ≥28 days from prior anti-cancer therapy, and women of child-bearing potential and men with partners of child-bearing potential must agree to practice sexual abstinence or to use contraception for 12 months following the last dose of IL-2 or until the first dose of the subsequent anti-cancer therapy.

2.2.2. Exclusion criteria

Participants meeting any of the following: simultaneously enrolled in any therapeutic clinical trial, concurrent use of other anti-neoplastic or investigational agents, psychiatric illness or social situation that would limit compliance with study requirements, pregnant or breastfeeding, active grade 3 or higher viral, bacterial, or fungal infection within 2 weeks, active major medical disorders of hematologic, cardiovascular, respiratory, or immune system, history of an organ allograft or prior cell transfer therapy, melanoma of uveal/ocular origin, hypersensitivity to study drugs, symptomatic and/or untreated brain metastases, left ventricular ejection fraction (LVEF) <45% or New York Heart Association (NYHA) functional classification Class > 1, and patients ≥60 years of age with any irreversible wall movement abnormalities on cardiac stress test, forced expiratory volume in 1 second (FEV1) of ≤60%, another primary malignancy within the previous 3 years (except for carcinoma in situ of the breast, cervix, or bladder; localized prostate cancer; and treated non-melanoma skin cancer), or received a live or attenuated vaccine within 28 days.

2.3. Interventions

2.3.1. Production and expansion of TIL

The manufacturing process begins at the clinical site with the surgical resection of a tumor lesion containing viable tumor material of ≥1.5 cm. Harvested tumor sample will be sent for centralized TIL (lifileucel) manufacturing. Autologous TILs are obtained from the tumor sample and expanded ex vivo through cell culture in the presence of the cytokine IL-2 and a monoclonal antibody (mAb) to human CD3. The final drug product is a cryopreserved live-cell suspension containing at least 1.8 × 10⁹ viable cells formulated for IV infusion. This dose was specified following the phase II registration trial (>1 × 10⁹ viable cells) and sponsor guidance at the time of protocol writing [8]. The total volume to be infused will be up to 500 mL, depending on the total cell dose. Lifileucel will be manufactured and supplied by Iovance Biotherapeutics.

2.3.2. Lymphodepletion

The patient will receive outpatient LD consisting of Cy 750 mg/m² IV in 250 ml normal saline over approximately 1 hour daily on days  −4,  −3,  −2, and Flu 30 mg/m² IV piggyback over approximately 15–30 minutes daily on days  −4,  −3,  −2,  −1.

2.3.3. TIL infusion

Patients will receive inpatient TIL infusion on day 0. Lifileucel will be transferred to the patient’s bedside, thawed, and administered via IV infusion at least 24 hours from the last dose of fludarabine. The initial infusion rate should be 1 mL/min for the first 5 minutes. The infusion rate may be increased to 5−10 mL/min, as tolerated.

2.3.4. IL-2 administration

The TIL infusion will be followed by high-dose IL-2 in the intensive care unit at a dose of 600,000 IU/kg IV every 8–12 hours for up to a maximum of six doses or as tolerated. The IL-2 infusion can begin as early as 3 hours and up to 24 hours after the completion of the TIL infusion.

2.4. Assessments

Blood collections will be conducted at screening, tumor harvest, and days  −7, 0, +4, +7, +14, +28, +42, +84, +126, +180, and  +365. Tumor response assessments will be done at 6 weeks (day +42), 12 weeks (day +84), 18 weeks (day +126), 6 months (day +180), 9 months (day +270), and 1 year (day +365). Blood TCR sequencing will occur to ascertain the persistence and diversity of T cell clones compared to lifileucel. A tumor biopsy will be obtained on day 42 to ascertain the persistence and diversity of TIL in the tumor compared to the procurement sample. Information on adverse events and serious adverse events will be collected after initiation of tumor harvest and study treatment and ends 6 months following the last administration of IL-2 dosing or until the start of a new anti-cancer therapy, whichever is earlier (all grades and any attribution until day 28 post-infusion, followed by grade 3–4 of any attribution until 18 weeks, followed by only grade 3–4 attributed to study drugs until 6 months).

2.5. Outcomes

2.5.1. Primary endpoint

The primary endpoint will be the percentage of TCR populations shared between the TIL product and PBMCs at approximately day 42 post-infusion. The TCR population is calculated as the frequencies of unique CDR3 sequences.

2.5.2. Secondary endpoints

Efficacy: ORR, DOR, PFS, and OS at 1 year after TIL infusion.

Safety: treatment-emergent adverse events (TEAE), serious adverse events (SAE), rates of hematologic toxicity, including transfusion requirements and length of hospital stay, at 6 months after TIL infusion.

2.5.3. Exploratory endpoints

Blood and tumor samples will be banked for future correlative analyses, including flow cytometry, next-generation sequencing, immunogenomics, and RNA sequencing, to characterize the immunome and microenvironment. We will also explore the duration of cytopenias, length of hospital stay, and immune reconstitution.

2.6. Participant timeline

The study is expected to complete accrual in 1 year. The end of the study is defined as when the last enrolled participant completes a pre-specified 1-year study follow-up.

2.7. Sample size

No formal sample size calculation or power analyses were conducted for this pilot study. This pilot trial will include 12 patients receiving TIL therapy with RD-NMA-LD, and 8 of the 12 planned patients have been treated as of December 2024.

2.8. Recruitment

Eligible participants will be registered through the KUCC Clinical Research Office’s central registration process. Written informed consent will be obtained prior to the performance of any study-related procedures or assessments.

2.9. Data collection, management, and analysis

2.9.1. Data collection methods

The Velos electronic data capture program will be used in this study. Electronic case report forms (eCRFs) will be completed for each participant enrolled and registered in this study. All CRFs will be customized per this study to emphasize completeness and accuracy. The investigatory and trained study staff will enter and edit the data via a secure network with secure identification and password requirements. A complete electronic audit trail will be maintained. Source documents, including medical charts and any other clinical worksheets, pathology, procedural, and imaging reports, serve as evidence of the existence of the participant and the data collected for this trial. Data captured in the eCRF must be transcribed from source documents and consistent with any discrepancies explained and documented.

2.9.2. Data management

All eCRFs and resulting data will be developed and maintained per the pertinent regulations and guidelines for using computerized systems in clinical trials. All users of the eCRF system will be trained prior to using it. A risk-based monitoring approach will focus on critical variables and triggered events and ensure the eCRF accurately reflects data recorded in source documents.

2.9.3. Statistical methods

Descriptive statistics will be used to describe the baseline characteristics of the data. The primary outcome of interest, the percentage of the Tumor-infiltrating lymphocytes (TIL) product at approximately day 42, will be summarized concerning mean and standard deviation (SD). Using the Clopper-Pearson Exact method, the ORR will be presented as a percentage with a 95% confidence interval (CI). The secondary outcomes, TEAE, SAE, and rates of hematologic toxicity will be presented as percentages with 95% CI. The DOR and length of hospital stay will be summarized with respect to mean and SD. The OS and PFS will be summarized using Kaplan−Meier curves and estimation of medians with 95% CI. Safety endpoint data sets are summarized descriptively. Correlative analyses will be carried out using Spearman’s correlation and chi-square analyses. All the analyses will be carried out using Statistical software SAS version 9.4 or R version 3.6.1.

2.10. Monitoring

2.10.1. Data monitoring

The KUCC Clinical Site Managers will regularly monitor data to comply with good clinical practice (GCP). The study will be monitored at appropriate intervals, no less than those assigned per the KUCC protocol review and monitoring committee (PRMC) risk level designation, to assure compliance with GCP and to assess the data quality and study integrity. Review of the case report forms, cross-reference with source documents, and review of trial-related regulatory documents and logs will be monitored following the risk-based approach outlined in the monitoring standard operating procedures. The study monitor will ensure the trial is conducted according to protocol design and regulatory requirements. The monitor will have access to all relevant study documentation and source data for data verification. The monitor will complete a follow-up letter and provide it to the sponsor investigator.

2.10.2. Harms

The KUCC Data Safety and Monitoring Committee (DSMC) will monitor every participant receiving treatment on this protocol for the safety, conduct, and scientific progress of research protocols and the validity and integrity of the data for clinical trials. This protocol will adhere to the policies of the currently approved KUCC Data and Safety Monitoring Plan, according to the NCI and the KUCC-Institutional Review Board (IRB) policy and guidelines. An overall assessment of toxicities as described in the protocol is reviewed at DSMC meetings. This review enables DSMC committee members to assess whether significant risks are occurring that would warrant study suspension, termination, or protocol amendment.

2.10.3. Stopping rules

Any death occurring within 60 days after infusion of the product will halt further accrual pending KUCC DSMC review. The study will continue only with KUCC DSMC’s determination that the event was unrelated to study procedures or drug products. Grade 4–5 toxicity, except expected grade 4 toxicity due to high-dose interleukin 2, related to the study drug, will halt further accrual pending KUCC DSMC review. The study may be stopped following a review of the safety data by the University of Kansas Medical Center according to the institutional standard procedures.

2.10.4. Auditing

The KUCC Quality Assurance Department will audit the study at appropriate intervals, no less than those assigned per the KUCC PRMC risk level designation, to assure compliance with GCP and Good Documentation Practices.

2.11. Ethics and dissemination

The study was approved by the KUCC Institutional Review Board (Study #00150697, IND #29694, Protocol #IIT-2022-LDTIL). Written informed consent will be obtained from the participants. The study will follow the ethical principles of the Declaration of Helsinki and adhere to the study protocol and applicable Good Clinical Practices. Informed consent will be obtained from all patients before any study-related procedures are conducted. The Sponsor/Investigator is primarily responsible for disseminating research findings and publications and will provide the funder with the opportunity to review and comment on any publications.

3. Conclusion

Tumor-infiltrating lymphocyte therapy (lifileucel) with traditional lymphodepletion has demonstrated durable responses in advanced melanoma patients who have limited treatment options. We hypothesize that a reduced dose of fludarabine and cyclophosphamide lymphodepletion will have similar TIL expansion and persistence and may lead to similar efficacy with a reduced toxicity profile with RD-NMA-LD compared to traditional NMA-LD.

Funding Statement

This investigator-initiated trial was funded by Iovance Biotherapeutics. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Article highlights

Background:

• Metastatic melanoma (MM) patients who have progressed following immune checkpoint inhibitors (ICI; anti-PD-1/CTLA-4 therapy) and targeted agents (BRAF/MEK inhibitors) have a dismal prognosis.

• In 153 mm patients previously treated with ICI and BRAF/MEK inhibitors, autologous tumor-infiltrating lymphocytes (TIL) therapy (LN-144, lifileucel) with non-myeloablative lymphodepletion (LD), using fludarabine (Flu) 25 mg/m² for 5 days and cyclophosphamide (Cy) 60 mg/kg for 2 days, and high-dose interleukin (IL)-2 showed an overall response rate (ORR) of 31% (n = 48). The median duration of response (DOR) was not reached after 4-year follow-up, and 4-year overall survival was 22%. Grade 3 or higher treatment-related adverse events were related to lymphodepletion and IL-2.

• We hypothesize that a reduced dose of Flu/Cy LD for TIL therapy will have similar TIL expansion and persistence compared to traditional Flu/Cy LD currently in use with TIL therapy and may lead to similar efficacy with a reduced toxicity profile with RD-NMA-LD as compared to traditional NMA-LD.

Methods:

• Study NCT06151847 is a single-center, open-label phase II pilot trial that evaluates the efficacy, in vivo persistence, and safety of TIL therapy following a reduced-dose conditioning regimen of Flu/Cy LD.

• Key inclusion criteria consist of patients with unresectable or metastatic melanoma (stage IIIC-IV) who have experienced disease progression after one or more lines of therapy.

• As of December 2024, 8 out of the 12 planned patients have been enrolled.

• Central TIL manufacturing requires a tumor specimen of at least 1.5 cm and involves ex vivo expansion in the presence of interleukin-2 (IL-2) and an anti-CD3 monoclonal antibody.

• Patients will receive the outpatient reduced-dose Flu/Cy LD regimen, consisting of Flu at 30 mg/m² on days  −4,  −3,  −2, and Cy at 750 mg/m² on days  −4,  −3,  −2. TIL infusion will be administered on day 0, followed by high-dose IL-2 at 600,000 IU/kg for up to six doses.

• The primary objective of the study is to determine TIL persistence using T-cell receptor (TCR) sequencing on day 42.

• The secondary objectives include evaluating efficacy parameters such as overall response rate (ORR), duration of response (DOR), progression-free survival (PFS), overall survival (OS), and the safety profile of TIL in conjunction with the reduced-dose Flu/Cy LD regimen.

• Exploratory objectives will focus on analyses that characterize the immunome and tumor microenvironment.

Future Directions for Research:

• If TIL persistence is demonstrated to be comparable between the reduced and high-dose Flu/Cy lymphodepletion regimens, we may consider conducting a larger trial using the reduced-dose Flu/Cy LD for TIL therapy in melanoma. The follow-up for the study will continue for 1 year.

Authors’ contributions

All authors contributed to the manuscript and fulfilled criteria per the uniform requirements set forth by the International Committee of Medical Journal Editors (ICJME) guidelines. All authors have reviewed and approved the final version of the manuscript.

Disclosure statement

The investigators have research funding from Iovance Biotherapeutics to conduct this investigator-initiated clinical trial. SHA has a consulting role with Incyte and research funding from CSL Behring and Miltenyi Biotec. JPM has consulting and advisory roles in Bristol Myers Squibb, Kite, Novartis, AlloVir, Envision, Autolus, Nektar Therapeutics, CRISPR Therapeutics, Caribou Bio, Sana Technologies, Legend Biotech, and Cargo Therapeutics. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.

Ethical disclosure statement

The authors declare that all research procedures were conducted in accordance with ethical guidelines. The study was approved by the University of Kansas Cancer Center Institutional Review Board (Study #00150697, IND #29694, Protocol #IIT-2022-LDTIL, version 3 dated April 02, 2024). Written informed consent from participants was obtained.

Data availability statement

The datasets generated and/or analyzed during the current study will be available from the corresponding author upon reasonable request. Data will be shared in accordance with institutional and sponsor guidelines and will require a data use agreement to ensure the confidentiality of participants.