What is TIL immunotherapy?

At its core, tumor-infiltrating lymphocyte (TIL) immunotherapy is the infusion of T cells cultured and expanded from a resected tumor deposit, in an attempt to amplify an individual’s ability to mount an immune response capable of eliminating established tumors. Conventional T lymphocytes equipped with billions of individual T-cell receptors (TCR) patrol the body constantly and surveil peptides bound to major histocompatibility complex molecules, the end product of degraded and processed intracellular proteins. Once a TCR is triggered by tumor-associated antigen, downstream signaling results in activation and proliferation of T cells, and the frequency of tumor-reactive T cells is thus generally much higher within tumors (“infiltrating”) compared with the peripheral blood.1–3 However, a plethora of immunosuppressive and immune-escape mechanisms facilitate the progression of metastatic cancers, despite T lymphocyte surveillance and tumor infiltration.4 The concept of TIL immunotherapy was first pursued in murine cancer models, and TIL expanded to large numbers ex vivo were effective at controlling metastatic tumors when adoptively transferred after lymphodepleting chemotherapy or radiation followed by interleukin-2.5 These basic principles have led to the development of TIL immunotherapy in humans, discussed in this article, where a highly personalized, and polyclonal, effector T cell product is manufactured from a freshly resected tumor and reinfused into patients with cancer as a living drug.

Major components

While immune checkpoint blockade pharmacologically targets immunosuppressive mechanisms in the tumor microenvironment to unleash the endogenous antitumor T cell response, TIL immunotherapy combines three main components: (1) it amplifies and activates T lymphocytes ex vivo in a way that cannot be achieved naturally in vivo, (2) it blunts immune suppressive mechanisms and increases homeostatic cytokines prior to TIL infusion by the administration of a short course of lymphodepleting chemotherapy, and (3) it supports the persistence and expansion of infused TIL with recombinant human interleukin-2 (IL-2, aldesleukin). Perhaps most importantly, the polyclonal nature of the TIL product more appropriately matches the dramatic heterogeneity of solid tumors when compared with the more targeted CAR approaches in more clonal hematological malignancies.

As shown in figure 1, TIL immunotherapy starts with the surgical harvest of a tumor of sufficient size, usually a metastasis. Activation and expansion of autologous TIL can take 3–6 weeks, depending on the manufacturing process. The treatment regimen consists of three components: non-myeloablative lymphodepletion (LD) of the patient, TIL infusion, and IL-2 supplementation. The general medical management of this treatment schema is summarized in figure 2.

Figure 1

TIL immunotherapy general scheme picturing the excision of a patient’s tumor, creation of a TIL product, and reinfusion back to the same patient. (A) The commercial model uses a centralized manufacturing process and delivery of a cryopreserved product to allow for greater geographical access to treatment. The point-of-care model can be similar, obviating the need for shipping. IL-2, OKT3 (a stimulatory anti-CD3 monoclonal antibody) and irradiated feeder cells are standard expansion conditions for TIL. (B) The local investigator model allows for ongoing translational research and manufacturing of a non-cryopreserved clinical product. Hypothesis-driven refinement of manufacturing techniques may include different approaches for TIL initiation (eg, separate individual fragment-based cultures vs a single culture derived from multiple fragments or tumor digest), methods of TIL expansion (eg, varying composition and concentration of cytokines or other adjuncts), and/or strategies to select and enrich for tumor-reactive TIL. MHC, major histocompatibility complex; TCR, T-cell receptor; TIL, tumor-infiltrating lymphocyte.

Figure 2

A generalized overview of the timeline, components, and clinical parameters associated with TIL immunotherapy. The timeline is in days, with TIL infusion on day 0. (A) After multidisciplinary discussion, the decision to pursue TIL begins with surgical tumor harvest for delivery to a manufacturing facility or research team. The time to product availability varies from 3–6 weeks. In some settings, this interval may be longer and warrant bridging therapy. The active treatment phase begins with lymphodepletion and lasts ~18 days with further marrow recovery following discharge. (B) The length of hospitalization depends in part on institutional guidelines, with inpatient care preferred until resolution of acute neutropenia. (C) Lymphodepleting chemotherapy usually consists of cyclophosphamide and fludarabine as shown. TIL are infused over 20–30 min on day 0. Some products may consist of multiple cryopreserved bags and are administered sequentially on day 0. IL-2 therapy can proceed later on day 0 after monitoring for acute symptoms related to the cell product administration. (D) The listed common side effects are likely to affect every patient and may range in severity. More critical side effects are less common. Rapid clinical recognition often leads to successful management, but fatalities have occurred (range 1%–7.5%).8 61 (E) Various clinical and laboratory parameters demonstrate the transient nature of many abnormalities. Weight loss of 5% over the course of therapy is not uncommon. All patients experience severe cytopenias, a feature of the lymphodepleting design rather than an unintended side effect of TIL/IL-2 infusions. (Patient level data).52 ALC, absolute lymphocyte count; ANC, absolute neutrophil count; BP, blood pressure; G-CSF, granulocyte-colony stimulating factor; HR, heart rate; TIL, tumor-infiltrating lymphocyte; WBC, white blood cell.

Figure 3

Common patient scenarios during IL-2 administration. (A) Patient A developed relative hypotension during the first day of IL-2 dosing with more severe hypotension after the fourth dose. The patient recovered with fluid hydration before delayed administration of the fifth dose. Dosing was ultimately stopped for persistent hypotension and tachycardia after the sixth dose. Low-grade tachycardia during the latter recovery period was associated with febrile episodes. Neutropenia resolved on day +9. (B) Patient B developed oliguria during IL-2 dosing initially treated with fluid bolus. Dosing was stopped for oliguria with rising creatinine. Diuretic use was initiated 24 hours after the last dose during the period of acute kidney injury. Creatinine resolved slowly without further intervention. potassium remained within normal limits throughout. (C) Patient C developed tachypnea and subjective shortness of breath after a sixth dose of IL-2, without immediate hypoxia. At onset of hypoxia, supplemental oxygen by nasal cannula was administered (shaded yellow) and diuretic therapy was started. Weight, oxygen saturation, and respiratory rate returned to pretreatment values. BP, blood pressure; DBP, diastolic blood pressure; JR, heart rate; HR, heart rate; SBP, systolic blood pressure.

General efficacy and first approvals

TIL immunotherapy has been demonstrated to mediate durable complete tumor regression and potential cure in patients with metastatic melanoma, even in patients with disease refractory to prior immune checkpoint therapy (table 1).

Table 1

TIL immunotherapy efficacy summary

After an initial first-in-human description in 1988, the landmark study of TIL immunotherapy associated with LD and IL-2 supplementation in patients with metastatic melanoma was published in 2002 by the team of Dr. Steven A. Rosenberg at the Surgery Branch of the US National Cancer Institute (NCI).6 76 7 Since then, hundreds of patients with diverse cancer types have been treated with TIL in multiple institutions. Proof of concept that TIL can be effective in multiple metastatic solid cancer types has been reported. The use of metastatic melanoma has been extensively studied and is the furthest in clinical development.

Table 1 summarizes the efficacy results of the largest prospective trials published using similar lymphodepleting regimen and high-dose IL-2 supplementation. The most mature prospective data from a single center have been reported in 224 patients with late-stage metastatic melanoma by the NCI, showing an objective response rate (ORR) of 51% and complete response (CR) rate of 22%.8 Importantly, patients with CR demonstrated a 10-year melanoma-specific survival of 96%. Furthermore, a meta-analysis including 410 patients from 13 studies published between 1988 and 2016 from 6 centers in the USA (including the NCI), Europe, and Israel revealed a pooled overall ORR of 41% with an overall CR rate of 12%, with similar durability of CR.9

The first randomized controlled trial was conducted in the Netherlands and Denmark to compare point-of-care manufactured TIL versus ipilimumab, in 168 patients with metastatic melanoma, 86% of whom had received prior anti-PD-1 therapy. The ORR was 49% including 20% CR in the TIL group, significantly higher than the immune checkpoint group.10 The median progression-free survival, the primary endpoint of the study, was 7.2 months in the TIL arm, compared with 3.1 months in the ipilimumab arm. The quality-of-life scores were similar in both groups, and cost-effectiveness analysis conducted by the Dutch Healthcare Authority was favorable for TIL.11 These results have led to early access programs for patients with metastatic melanoma who had received anti-PD-1-based therapy to receive point-of-care manufactured TIL since 2023 in Denmark and the Netherlands.

A commercial TIL product, lifileucel, is centrally manufactured from tumors harvested at individual centers, cryopreserved, then shipped back for administration to patients. Lifileucel was tested in sequential prospective single-arm cohorts totaling 153 patients with metastatic melanoma, pretreated with a median of three lines therapy. In these later stage patients, the ORR was 31.4% with 5.2% CR, and the median duration of response was not reached at a median study follow-up of 3 years.12 13 These results led to the Food and Drug Administration (FDA) approval of lifileucel for patients with melanoma after progression on anti-PD-1 therapy and on anti-BRAF with or without MEK inhibitors for patients with BRAFV600E mutated tumors in February 2024. Consensus guidelines were published at the time of commercial availability.14

These trials in melanoma have used methods that allow for the growth of all possible lymphocytes from a harvested tumor, or bulk-expanded TIL. As shown in table 1, these techniques have also been used for common epithelial cancers and have been associated with encouraging efficacy signals mainly in patients with late-stage non-small cell lung cancer and HPV-related malignancies such as cervical cancer.15 16 Larger, pivotal trials are currently being conducted in these indications to confirm efficacy.

The administration of bulk-expanded TIL in patients with gastrointestinal cancers has to date not been associated with objective responses.17–19 Immunological analysis of TIL generated from epithelial cancers have identified neoantigen-reactive TIL in up to 82.6% (62/75) of tested metastases derived from gastrointestinal cancers20 and 67% (29/43) derived from breast cancer.21 Proof of concept of the efficacy of neoantigen-enriched or selected TIL in these non-melanoma patients have been reported.21–24 The strategy of selected TIL administered with LD, IL-2 and pembrolizumab is currently being evaluated in an ongoing clinical trial. Multiple avenues are currently being explored by academic institutions and biopharmaceutical companies to better select and improve function of TIL aiming to increase response rates and translate clinical benefit to less immunogenic cancers.25

Factors associated with response to TIL immunotherapy

There are currently no validated predictive biomarkers of response or resistance to TIL immunotherapy. Correlative research has mainly focused on associations between the immune features of the TIL product and response to TIL immunotherapy in metastatic melanoma patients. A higher likelihood of responding to TIL has been associated with the total number of TIL and CD8+ TIL, or tumor-reactive TIL, infused,26 27 the proliferative potential of TIL infused (longer telomeres),28 the proportion of CD8+ TIL expressing CD27 (marker of less differentiated effector memory T cells)29 30 or of stem-like phenotype (CD39negCD69neg),31 the release of interferon-gamma (IFN-γ) in response to autologous fresh tumor stimulation,32 and CD8+ TIL reactivity to neoantigen peptides inferred from tumor sequencing.33 34 The persistence of infused TIL at 1 month has also been associated with higher likelihood of response.30 Tumor factors such as a high number of immunogenic neoantigens predicted from sequencing may be associated with response to TIL and longer survival.35 36 One deep analysis of a small cohort of patients suggested that cellular crosstalk in the harvested tumor may be important.37 During immune reconstitution after LD and TIL administration, immune suppression mediated by circulating regulatory T cells (Treg)38 and myeloid-derived suppressor cells (MDSC) could limit clinical response.39

Currently, however, none of these factors can be used to guide the clinical decision to pursue TIL for an individual patient. To this end, more immunological investigation is needed to understand the baseline characteristics of patients and tumors that are associated with response to TIL therapy. Although the highest response rates have been observed in patients with melanoma when TIL has been used in earlier lines of treatment, objective response still remains possible in heavily pretreated patients.

Expected side effects

Because TIL immunotherapy is composed of multiple elements, expected side effects are best discussed in the context of each component of therapy. The initial surgical component is intended to be of minimal risk to the patient with low rates of site-specific complications and infections, emphasizing the importance of patient selection and surgical technique.40–43 The subsequent treatment components and their toxicities are discussed more fully below, but in general, the regimen is associated with multiple acute toxicities that generally resolve with appropriate management within the hospitalization period although there are rare, serious and even fatal side effects can occur (figures 2 and 3).

Patient selection and medical oncology considerations

While TIL immunotherapy continues to be studied in patients with a wide variety of cancers, presently the greatest application will be in patients with metastatic melanoma after cessation of or progression through first-line checkpoint inhibition. With the recent FDA-approval and durable response rate in checkpoint-refractory disease, oncologists should consider the availability of TIL at their institution, within their referral network, or at regional cancer centers before beginning second-line therapy. For non-melanoma patients, access to TIL immunotherapy currently remains only through clinical trials. TIL therapy will not be appropriate for every patient, and multidisciplinary evaluation is key in guiding therapeutic decision-making.

Patient selection

Medical considerations in the selection of patients reflect the anticipated side effect profile of the regimen (eg, cytopenia following LD, hemodynamic fluctuations and fluid shifts with IL-2, as discussed below) and the pace of the patient’s disease progression (ie, practical concerns of scheduling, TIL manufacturing time and expected treatment date). Most importantly, consideration of TIL is only possible in the presence of at least one harvestable metastatic deposit measuring at least 1.5 cm in its largest diameter. Other metastases remaining after surgery can be used to assess treatment efficacy.

In the interest of patient safety during initial implementation of a new regimen, institutions or medical groups may choose to adhere to the historical protocol eligibility criteria as outlined in detail in consensus guidelines.14 As with all oncological therapy, patients should be fully aware of the nature of the treatment (surgical excision for tumor procurement followed by a rigorous one-time inpatient treatment), potential risks, and benefits associated with treatment. All patients will experience common, severe, but transient side effects, and ideally, patients can be counseled by the cell therapy treatment team for better understanding before committing to surgical harvest.

Patient management for treating oncologist

Once the decision to pursue TIL has been made, outpatient management is divided into presurgical, postsurgical (TIL manufacturing period), and post-TIL components. The degree of involvement of the treating oncologist during each of these phases depends on the structure of the TIL program at any given institution.

In the presurgical phase, considerations of the pace of disease may guide the timing of surgical harvest. Protocol-based TIL and standard guidelines generally required a treatment-free interval of ~4 weeks off systemic cytotoxic therapy prior to surgical resection. While there are laboratory and observational data supporting resection while on therapy, the clinical efficacy of TIL harvested during active treatment is unknown. For patients receiving periodic infusions (eg, monoclonal antibodies), a natural window can be created for resection. For patients on daily oral therapy (eg, MAPK pathway inhibitors), this may require more coordination and consideration. Of note, rapid tumor progression following cessation of BRAF/MEK inhibitor therapy is a well-recognized occurrence that should be taken into consideration when stopping these drugs prior to the harvest procedure. An interval of 4–6 weeks is generally required after anti-VEGF monoclonal antibodies that can compromise wound healing (eg, bevacizumab). Outside of medical considerations, scheduling a tumor harvest must also encompass the availability of the surgeon, the operating team, and manufacturing centers (local or central). If the treating oncologist believes a patient’s disease may progress rapidly or lead to important complications (such as progression of brain metastases) while waiting for tumor harvest, the treatment strategy should be extensively discussed within a multidisciplinary team as the patient may not be a good candidate for a TIL immunotherapy.

Ideally, most patients with melanoma will not require intervening oncological treatment during the 3–6 weeks of TIL manufacturing. However, as “bridging” therapy, clinicians may occasionally elect to continue a prior line of therapy or more rarely begin a next-line therapy for patients with rapidly progressing cancers or for participation in protocols with a prolonged manufacturing timeline. Any “bridging” treatment aimed at maintaining disease stability can be stopped when TIL immunotherapy is scheduled. A next-line therapy should likely continue to the point of maximal benefit before embarking on TIL immunotherapy. If a disease is present for which radiation is likely to be considered for palliation (ie, painful or unstable bone metastases) or treatment (brain metastases), radiation should be considered during the postsurgical window before administration of TIL to prevent irradiation of the transferred TIL in the peripheral circulation.

Tumor selection and surgical oncology considerations

Implementing TIL therapy requires a multidisciplinary team that includes surgical oncologists with various areas of expertise (thoracic, hepatobiliary, endocrine, etc). Plastic surgeons may also play a role depending on local practice patterns. Patients with metastatic cancer can only access TIL therapy when surgeon-stakeholders can coordinate timely, low-risk resections of an appropriate size metastasis(es), ensure proper handling of harvested tissue for TIL manufacturing, and employ best practices to expedite patient recovery sufficient for TIL immunotherapy. Occasionally, metastasis resection for symptom management may be indicated and surgeons should consider consultation with their clinical and/or research cell therapy team(s) as the resected tumor may be also useful for TIL manufacturing or research.

Patients fit to undergo TIL immunotherapy must also tolerate general or local anesthesia necessary to perform the least invasive possible surgery and should be free of medical issues that could hinder timely recovery during TIL manufacturing. Surgeons need to review the necessary washout time off systemic therapy to schedule the procedure. The following general considerations (highlighted in box 1) are provided to guide surgeons in their decision-making within the multidisciplinary team involved in TIL therapy.

Box 1 Surgical considerations of tumor harvest for tumor-infiltrating lymphocyte (TIL)

Assessment

Targeted metastasis defined jointly by the cell therapy and surgical teams

≥1.5 cm metastasis, non-necrotic and non-cystic

Can be resected with minimally invasive technique

Avoid intraluminal bowel/bronchial/mucosal tumors

Scheduling

Define chemotherapy washout time and optimal date of surgery

Book additional preoperative testing and confirm operating room availability

Confirm manufacturing availability (locally or centrally) for receipt of specimen

Resection

OR staff trained and aware of specific procedures

Shipment logistics are established; transport media is prepared and available

Surgeon performs metastasectomy with minimal margins to minimize morbidity

Prosection

Maintain sterility similar to organ procurement

Performed in operating room at back table by surgeon or under aseptic conditions by a trained pathologist or technician

A small piece of tumor tissue is sent to pathology to confirm the presence of cancer (frozen section may be used)

Normal tissue is trimmed from the tumor.

Bulk of the remaining tumor is placed in transport media in preidentified container (can be divided into smaller pieces if necessary)

Handling and shipment

Parafilm-sealed container is placed in double layer plastic bags before transport (final container varies based on destination)

Tissue procurement and transportation forms are signed

Post operative period

Tumor selection

Typical patients bear multiple metastases, and tumor(s) to be harvested for TIL manufacturing should be clearly identified by a surgeon as part of the multidisciplinary TIL therapy team. The main factor associated with successful TIL manufacturing is the initial amount of viable tumor tissue. A minimum tumor greatest diameter of 1.5 cm, or approximately 2 cm3 in volume, is generally sufficient for successful TIL manufacturing. TIL therapy has been expanded from a variety of tumor anatomical sites, most commonly subcutaneous tumors and lymph node metastases, but also from other sites including the lungs, liver, and the adrenal glands.41 There is no definitive evidence that a specific metastatic site yields better TIL or is associated with higher response rate. Practically, tumors in contact with high bacterial content structures, such as the bowel, the bronchus, mucosal surfaces, or the liver in patient with stented bile ducts, carry an increased risk of bacterial contamination during TIL manufacturing and should be avoided. If TIL is being delivered in the context of a clinical trial, metastatic deposits selected by the team for treatment response evaluation should be clearly identified and distinguished from the lesion(s) to be harvested.

Excisional surgical biopsy

The goal of the procedure is to procure sufficient tumor for TIL manufacturing favoring the least invasive technique and lowest impact on healthy tissue so as to hasten patient recovery. Same-day outpatient procedures are ideal, but the patient may need short-term hospitalization. In contrast with conventional curative intent surgery, enucleation and close/positive margins are acceptable. Partial excisions may be necessary to avoid excess surgical morbidity but should be avoided when possible. Conventional surgical instruments are used according to surgeon preference. All efforts to reduce postoperative complications should be employed to prevent delay of cell therapy. At present, manufacturing TIL from core biopsies should only be done under research protocols. Low tumor input material likely increases manufacturing failure rates, reduces TIL diversity, and reduces end-product TIL numbers, which altogether may reduce TIL therapy efficacy.

Tumor handling

Surgeons need to be supported by dedicated staff for logistics and documentation, with clear procedures and excellent chains of communication and custody to maximize the chance of successful TIL manufacturing. The operating room and pathology staff should be aware that the intent of the operation is tumor procurement for TIL manufacturing, not curative resection, and be trained on appropriate procedures and necessary documentation, given that the greatest part of the surgical specimen will not go to pathology. Preoperative communication with the anesthesia team is critical to avoid the use of steroids as part of the induction regimen.

Sterility must not be breached, as tumors are used to manufacture a therapeutic cell product. The excised tumor should be handled with a similar mindset as organ procurement for transplantation. If tumor procurement is part of a larger clinical procedure, best practices would prioritize tissue harvest before potential contamination of the surgical field.

At the preoperative time out, the following TIL-specific matters should be reviewed in addition to the standard-of-care checklist:

Verify if all tumor-procurement and transportation logistics forms are available. Personnel responsible for related documentation and obtaining signatures should be clearly identified.

Confirm which tumor will be removed and the data needed to be documented (typically the resected organ, time at which the tumor is resected, macroscopic tumor measures, time at which tumor is placed in transportation media).

Confirm who will perform the tumor prosection (trimming) and how it will be done: Conventionally, a small tumor piece is sent to the pathology department for standard analysis (histological confirmation, tissue features, ±immunohistochemical staining). Frozen section may be used if there is an immediate concern about the tissue but should not delay processing. Nearly all of the resected tumor will be sent for TIL manufacturing. Commercial protocols require normal tissue to be trimmed off the tumor, with best efforts to provide only gross tumor for manufacturing. Larger tumors may be cut into smaller pieces, per protocols, usually not less than 1.5 cm in diameter. Surgeons are best positioned to process tumor efficiently immediately after resection on a back table within sterile fields using a fresh blade. Some institutions may prefer to have trained technicians and pathologists for this task performed in aseptic conditions outside the surgical suite.

Confirm transportation material and logistics: The harvested tumor (or tumor pieces) is sent for TIL manufacturing in preidentified manufacturing grade sterile container(s) filled with a defined volume of transportation media. Transportation media may be supplemented by antibiotics and antifungal agents at specific concentrations typically on the day of the surgery by dedicated staff, with appropriate documentation. The procured tumor is placed in the appropriate container and typically leaves the operating room within two biohazard bags, put into a preconditioned, temperature-controlled (4°C) transportation device to slow metabolism and reduce cell loss. The cell manufacturing laboratory may be within the institution (point-of-care manufacturing) and transported per internal procedure, or in an outside, centralized laboratory, requiring dedicated, prearranged courier. Standard institutional perioperative practices for pathology submission should be applied to the portion of the specimen sent for frozen section and/or final pathology. In research institutions, additional tissue not used for pathology or TIL manufacturing may be used to support local research or biospecimen banking if consistent with informed consent and local research practices.

Postoperative care

Surgeons are likely to need to reassess patients postoperatively to ensure they have sufficiently recovered from the procedure and take part in multidisciplinary decisions on when to initiate LD for TIL infusion. The final pathology should be reviewed. Meanwhile, patients need to have access to their surgeon and/or the clinical treatment TIL team in case of postoperative complications. Any complications that might delay treatment should be communicated promptly and documented appropriately.

TIL immunotherapy treatment

TIL immunotherapy requires coordination of several clinical elements: non-myeloablative LD, TIL delivery/thawing (if cryopreserved) and infusion, IL-2 administration, the concomitant delivery of medications necessary for symptom prophylaxis or management, and the availability of specific emergency medications. The design of the TIL regimen was optimized over decades of sequential, empirical clinical trials in patients with metastatic melanoma, primarily seeking to maximize oncological benefit but also refining the approach for patient comfort and ease of administration. The treatment regimen is intended to be an uninterrupted sequence of events scheduled around the expected availability of the cell product which will be infused on site as a fresh or cryopreserved product. Depending on each center’s resources, schedules for administration of cryopreserved TIL products are likely best based around a cell infusion plan that allows for early- to mid-week management of the most potentially toxic components of therapy (cells and IL-2). Figure 2 summarizes key clinical parameters of TIL immunotherapy discussed below, and online supplemental table 1 summarizes typical pharmacological management in which supportive medications can be adjusted to reflect local hospital guidelines. Detailed checklists and nursing considerations have been previously published.14

Preparative LD with chemotherapy

LD was demonstrated to be necessary for the development of durable tumor regression in sequential trials involving patients with metastatic melanoma.30 44 45 The standard recommended non-myeloablative LD regimen consists of 2 days of cyclophosphamide (60 mg/kg daily) and 5 days of fludarabine (25 mg/m2 daily) (figure 2C). The regimen was designed to induce profound leukopenia and lymphopenia prior to cell infusion, with reduction of regulatory T cells and MDSCs, and an increase in homeostatic cytokines that promote T cell proliferation.28 46 While the depth and duration of cytopenia may vary with the patient’s treatment history, neutropenia, thrombocytopenia, and anemia should be expected. Lower doses of cyclophosphamide have been used to reduce toxicity, but in numbers too small to demonstrate similar oncological outcomes. In patients with epithelial cancers, a reduction in cyclophosphamide (30 mg/kg daily×2 doses) was associated with a slightly shorter duration of neutropenia and lower incidence of thrombocytopenia, but achieved the desired lymphopenia prior to TIL infusion (unpublished, Goff). It is also common to concurrently administer cyclophosphamide and fludarabine on the first two days, followed by 3 days of fludarabine, which shortens the LD schema from 7 to 5 days. Neutropenia should be supported with filgrastim or biosimilar granulocyte-colony stimulating factor (G-CSF); the most common practice is to start G-CSF administration the day after TIL infusion (figure 2E). The impact of G-CSF administration on either therapeutic response or prevention of sepsis has not been carefully studied.

Nausea is a common side effect of cyclophosphamide, and scheduled antiemetic prophylaxis can be beneficial. However, institutional protocols should be modified to avoid the use of steroids during TIL therapy. While rare, the most serious potential toxicity with high-dose cyclophosphamide is hemorrhagic cystitis. In most centers, this risk is minimized with preinfusion intravenous hydration to increase diuresis and administration of mesna (detoxifying bladder accumulation of acrolein). In selected patients, diuretics may be necessary to maintain a brisk urine output. For example, the NCI-Surgery Branch protocol requires 6 hours of hydration (0.9% NaCl+20 mEq KCl @ 1.5–2 mL/kg/hour), administers simultaneous mesna with each dose (15 mg/kg over 1 hour each day) followed by a mesna infusion (3 mg/kg/hour×23 hours), and monitors urine output every 2–4 hours (goal >1 mL/kg/hour). In contrast, fludarabine is well tolerated with minimal acute side effects and very rare long-term toxicity as described in the package insert. While practices may vary, the patient’s fluid status should be optimized and approaching euvolemia prior to cell infusion.

Whether non-myeloablative LD should be performed in an inpatient or outpatient setting depends on the hospital setting and local lymphodepleting protocols used in stem cell transplant and CAR T cell therapy. To prevent cyclophosphamide toxicities, a combination of intravenous bolus and oral administration can allow for outpatient administration.

Blood product transfusion

Only irradiated blood products should be administered to lymphodepleted patients. As a general goal, hemoglobin should be maintained greater than 8 g/dL with red blood cell transfusion. To mitigate the risk of spontaneous bleeding, platelet transfusions should be administered to keep levels of at least 10–20 K/µL, or 30 K/µL in patients who are at higher risk of bleeding (based on location of metastases, history of GI bleed, septic state, etc) or per institutional guidelines. It is recommended to avoid transfusion on the day of TIL infusion to avoid pulmonary congestion.

Vascular access

There is no consensus on the best catheter to use for TIL immunotherapy. The goals should include central vascular access for infusion of lymphodepleting chemotherapy, cells and IL-2, large bore access for potential fluid resuscitation, and consideration of pre-existing vascular central access. Common practice has been to infuse cells in temporary tunneled dual-lumen catheters placed by interventional radiologists into the internal jugular vein. TIL can be successfully infused through conventional central lines, sometimes in conjunction with supplemental large-bore peripheral intravenous access, or with multiple-lumen peripherally inserted central catheters (PICC lines). Though PICC lines may require an infusion pump rather than relying on gravity infusion, both practices have been successfully used to deliver TIL products. Best practices would reserve one lumen solely for cell infusion, remove conventional central lines when the risk of infection becomes significant (usually >7 days), and take into consideration that IL-2 is resuspended in 5% dextrose in water and not compatible with normal saline. Lines may be closed with citrate lock rather than standard heparin lock to mitigate the risk of heparin-induced thrombocytopenia (HIT), which is difficult to diagnose in lymphodepleted patients, but there is no comparative data to favor one approach over the other.

TIL infusion

Ideally, the TIL infusion product is administered 24–96 hours after the completion of the final dose of lymphodepleting chemotherapy. The beginning of this window allows for renal excretion of fludarabine (half-life 8–10 hours) to avoid potential harmful effects on the infused T cells. The end of the window signifies the beginning of marrow recovery and the loss of the potential benefits of lymphopenia and increased endogenous cytokines.

TIL infusion should occur in the inpatient setting with appropriate resources for critical care support if needed. In preparation, patients may be admitted in advance for establishment of vascular access and administration of prophylactic premedications (figure 2B). In advance of cell infusion, patients should receive gentle hydration (eg, 50 cc/hour) and premedications designed to reduce the risk of allergic reaction (eg, diphenhydramine). Typically, standing acetaminophen, indomethacin and antiemetics are initiated prior to TIL infusion to prevent the side effects of low dose IL-2 remaining in the infusion bag(s), and because intravenous IL-2 will be initiated within 4h–24h after TIL infusion (see below).

The TIL infusion product volume and appearance may differ based on the source and/or manufacturer. Investigational products in support of local research questions may be unique to each institution. Current commercial products are shipped as 1–4 frozen infusion bags each holding as much as 125 mL of product, with 5%–10% dimethyl sulfoxide (DMSO) as a cryopreservation excipient and may contain low dose IL-2. Local institutional guidelines for thawing cryopreserved cell products in qualified water baths, at the bedside or a nearby laboratory, are used to administer cells as early as possible after thawing to reduce the harmful effect of DMSO on T cells. If multiple bags will be administered, they are thawed and administered sequentially. The product can be infused via gravity or by pump, per institutional guidelines, in a line that has been primed with normal saline. For obvious reasons, leucocyte filters are not used on these infusion lines. An initial slow infusion rate (1 mL/min×5 min) is recommended for observation prior to increasing to a standard rate (5–10 mL/min).

While rare, hypersensitivity reactions may occur during initial infusion, again highlighting the need for the appropriate hospital setting and available emergency resources. More commonly, patients may experience transient dyspnea and/or hypoxia as the cells enter the pulmonary vasculature and should be monitored for resolution prior to the IL-2 phase of treatment. Severe chills and rigors may occur and can be treated with meperidine (25–50 mg) if not otherwise contraindicated. Unique to cryopreserved products, DMSO can cause patients to experience unpleasant taste and smell as it is processed and exhaled. DMSO can also cause a “warmth” sensation during infusion that resolves without intervention.

There is no known effective dose of cells that can predict likelihood of clinical response on an individual basis. In the most recent era, cell doses have ranged from 1×109–150×109 cells.10 47 The median number of infused cells most recent melanoma trials was 21.1×109 cryopreserved TIL13 and 40.9×109 fresh TIL.10

Interleukin-2

The final phase of TIL therapy is the intravenous administration of high-dose aldesleukin (600,000–720,000 IU/kg), the recombinant form of interleukin-2 (IL-2). IL-2 was initially investigated as monotherapy and received FDA approval for use in patients with metastatic melanoma and metastatic renal cell carcinoma prior to the era of immune checkpoint blockade and small molecule inhibitors. IL-2 provides non-specific stimulation of the patient’s T cell repertoire and is used in TIL therapy to promote proliferation and ongoing activation of the transferred cells. Each patient will respond differently to IL-2, and treating clinicians should be familiar with the assessment and mitigation of a wide range of potential side effects.48 While consensus guidelines have been written for single agent IL-2 administration aiming to deliver up to 18 doses across multiple cycles,49 a key difference for consideration by TIL clinicians is single cycle administration as a supportive medication. In the most recent study that allowed dosing to tolerance, a median of 4 doses was administered.10 Additionally, the lymphodepleted status of the patients requires prompt assessment to distinguish IL-2-related symptoms from possible neutropenic infections. Since side effects are cumulative, understanding when to withhold or stop IL-2, considering multiple parameters, is probably the most critical knowledge to gain for safe administration (see box 2 for general IL-2 stopping criteria, Ref 14 for expanded criteria).

Box 2 General IL-2 stopping criteria

Immediate

Somnolence requiring intubation for airway protection

Confusion/hallucination

Myocardial infarct

Sepsis

Persistent hypoxia (>4L/min oxygen)

Arrythmias

Anuria

Persistent diarrhea (after medical management)

Delay: (treat/monitor for resolution)

Hypoxia (supplemental oxygen via nasal cannula)

Hypotension and tachycardia (±fluid)

Elevated transaminases (>5×upper limit of normal)

Hyperbilirubinemia (>5×upper limit of normal)

Oliguria (intravenous fluid)

Fatigue

If 24 hours elapse without significant recovery, IL-2 should be discontinued.

A combination of multiple symptoms should lower the threshold to stop IL-2.

Since IL-2 renders patients more susceptible to gram-positive cocci infection by inducing a chemotactic defect in neutrophils,50 antibacterial prophylaxis covering these organisms, such as oxacillin or cefazolin, is administered until 24 hours after the last IL-2 dose, in addition to infection prophylaxis covering the lymphodepleted state.

The first IL-2 dose is generally administered within 4–24 hours after TIL infusion. Post-TIL infusion respiratory symptoms should be resolved, blood pressure should be normal, and a baseline assessment of mental status should be performed. IL-2 is then administered every 8–12 hours, generally for a maximum of six doses in current practice. In experienced centers, IL-2 is administered on a standard inpatient unit, generally with access to telemetry support and a favorable nurse-to-patient ratio. Vital signs and urinary output are monitored every 4 hours, with the possibility to shorten the interval as needed. Daily weight is recorded, and routine labs (eg, complete blood count, basic metabolic panel) are performed at least daily. Programs building expertise may elect to dose patients in a step-down (medium acuity) or intensive care unit (ICU) for appropriate monitoring.

Peak effects are generally seen 4 hours after administration. Common side effects can be treated in a directed fashion including fevers (anti-pyretics), chills (hydromorphone, meperidine), pruritus (anti-histamines), nausea/emesis (anti-emetics except for steroids), diarrhea (loperamide, etc.), and dyspnea/hypoxia (supplemental oxygen).14 51 After each dose, mild hypotension resulting from a combination of vasodilation and IL-2-induced capillary leak is expected, as well as compensatory mild tachycardia. This is initially treated with judicious fluid resuscitation. Persistent or refractory tachycardia should be evaluated with ECG, and IL-2 should be stopped for arrythmia. Expected impact on renal function and body weight are summarized in figure 2E. Decreasing urine output may accompany hypotensive episodes but may also occur as an independent side effect of IL-2. Creatinine may rise with cumulative dosing. Mild edema is common, as patient weight typically increases along IL-2 administration and fluid administration. Hypoxia most often results from low-grade pulmonary edema, which is diagnosed on chest X-ray in approximately 25%–30% of patients.10 47 Severe hypoxia should prompt urgent investigation to rule out other diagnoses (inflammatory or infectious). The use of diuretics is commonly initiated 24–48 hours after the last dose of IL-2 in stable patients, aiming for recovery of baseline weight; earlier administration may lead to significant hypotension. Confusion, vivid dreams, and hallucinations are manifestations of IL-2 neurological toxicity that should prompt cessation of therapy. Some common patient scenarios are illustrated in figure 3.

Patients should be reassessed prior to every dose (every 8–12 hours); doses may be skipped if serious symptoms are not resolved. If 24 hours elapse without significant recovery, IL-2 should be discontinued. Up to 10% of patients may require ICU admission per institutional practice for low-dose pressor and non-invasive ventilatory support such as high-flow oxygen via nasal canula. Patients and their families should be counseled that the effects of IL-2 can continue beyond cessation of therapy (eg, creatinine may continue to rise, confusion may worsen), but