Evidence for lymphodepleting preparative regimens

The necessity of temporary lymphodepleting preconditioning before TIL infusion remains an important aspect in ACT with TIL. The first evidence for the need of lymphodepletion with either chemotherapy or total body irradiation (TBI) was demonstrated in murine models, where improved response rates were seen with TIL after lymphodepletion [20, 21]. Lymphodepletion with either TBI or NMA chemotherapy is thought to improve the effector function of TIL in several ways. Firstly, data from several studies suggest that the endogenous subpopulation of CD4⁺CD25⁺ regulatory T cells (Tregs) capable of suppressing immune responses may be depleted [22]. Secondly, lymphodepletion of the host reduces the pool of endogenous lymphocytes competing with the transferred T cells for homeostatic cytokines, especially IL-7 and IL-15 [23]. These cytokines are produced by non-lymphoid sources in response to lymphopenia, where IL-7 is required for the proliferation and survival of T cells and IL-15 maintains and improves the proliferation of the T cells [24, 25]. Lastly, lymphodepletion is believed to generate “physical space” for the infusion product.

In 2002, the Surgery Branch of the NIH demonstrated the clinical importance of lymphodepletion before TIL infusion. In this study, 13 patients with metastatic melanoma were treated with cyclophosphamide 60 mg/kg/day for 2 days and fludarabine 25 mg/m²/day for 5 days prior to TIL infusion and achieved an ORR of 46% [7], which compared favorable to response rates of 31% without prior lymphodepletion [6]. In 2008, this same group examined the effect of intensifying the lymphodepleting regimen by adding TBI to the above mentioned NMA chemotherapy and improved clinical outcomes with this strategy. Patients were treated with cyclophosphamide and fludarabine with addition of either 2 Gy or 12 Gy TBI, with 25 patients in each cohort. Compared to the cohort treated solely with chemotherapy showing an ORR of 49%, addition of TBI with 2 Gy or 12 Gy improved these ORR to 52 and 72% respectively [16].

In a follow-up randomized trial, the additional benefit described above of TBI in addition to NMA chemotherapy for the ORR could not be confirmed. A total of 101 patients with metastatic melanoma were either treated with NMA chemotherapy as described above per standard protocol, or in combination with 1200 cGy TBI (TBI 2 Gy twice a day for 3 days) prior to TIL infusion. Clinical outcome was similar in both treatment arms, with durable CR seen in 24% of patients in both cohorts and no significant difference in ORR of 45 and 62% in patients pretreated with NMA chemotherapy alone or with the addition of TBI respectively (p = 0.11). Of note, addition of TBI did result in additional toxicity, namely thrombotic microangiopathy in 27% of patients [26].

Temporary lymphodepletion with chemotherapy, TBI or a combination thereof appear to have an additive effect on the efficacy of TIL therapy as described above. Nevertheless, the question remains what the most optimal regimen is, in terms of both duration and depth of lymphodepletion and in terms of which drug(s) to use. Answers to these questions are not only relevant to further improve response rate to TIL therapy, but also to minimize toxicities, now predominantly consisting of transient pancytopenia and febrile neutropenia occurring in 37–96% of patients [18, 19].

To address these questions, the Sheba Medical Center, Israel, is currently conducting a phase II clinical trial exploring the efficacy of reduced intensity lymphodepletion using fludarabine 25 mg/m² for 3 days (instead of five per standard protocol and no addition of cyclophosphamide) followed by TBI (2 Gy single treatment for 1 day prior to TIL infusion (NCT03166397). This clinical trial is still recruiting and is expected to give more insight into the optimal lymphodepleting regimen prior to the infusion of TIL in melanoma patients.

The role of interleukin-2 in the current treatment protocol

Single agent IL-2 has received approval by the US Food and Drug Administration for the treatment of metastatic renal cell cancer and metastatic melanoma in 1992 [27] and 1998 [28] respectively. When used in combination with TIL, IL-2 is thought to enhance the anti-tumor response by continuous support of growth and activity of the infused TIL products. Studies suggest that IL-2 may enhance the inherent antitumor activity of CD8⁺ T cells and the cytolytic function of natural killer cells [29]. However, IL-2 is also associated with a variety of toxicities, some associated with capillary leak syndrome presented by edema, hypotension and reduced urine output within hours of infusion, but also fevers, rigors, myalgia and nausea. Most of these toxicities can be managed well by supportive measures [28]. However, so far no clear correlation between the number of IL-2 infusions and clinical response could be demonstrated. It is therefore worthwhile to reconsider the role of HD IL-2 administration in combination with TIL infusion.

A phase I trial at the NIH evaluated the anti-tumor effect of TIL therapy with varying IL-2 doses ranging from 0 to 720,000 IU/kg in 15 patients with metastatic melanoma. Patients receiving either low-dose (LD) IL-2 (72,000 IU/kg i.v. every 8 h up to 15 doses) (n = 3) or HD IL-2 (720,000 IU/kg i.v. every 8 h up to 12 doses) (n = 6) following NMA chemotherapy and infusion of TIL showed reduction in tumor volume. This effect was not seen in patients that did not receive any IL-2 (n = 6) [30]. Of importance, however, is that these findings are based on a small study and confirmation of these data would require a larger prospective trial. The CCIT, Herlev, Denmark, demonstrated clinical responses in patients with metastatic melanoma treated with lymphodepleting chemotherapy and TIL infusion followed by subcutaneous (s.c.) LD IL-2 injections (2 MIU for 14 days). Durable objective responses were seen in 2/6 (33%) patients and 2/6 (33%) patients showed disease stabilization [31]. In another phase I/II study by the same group, administration of intravenous IL-2 in a decrescendo regime also showed clinically significant responses with an ORR of 42%. In this trial, 25 patients with metastatic melanoma were treated with standard lymphodepleting chemotherapy and TIL infusion followed by 5 days of continuous infusion of IL-2 in a decrescendo manner, with 18 MIU/m² over 6, 12 and 24 h followed by 4.5 MIU/m² over 24 h for 3 days [18]. These data from the NIH and CCIT suggest that it might be possible to lower the dose of IL-2, without negatively effecting clinical outcome.

Currently, several clinical trials are being conducted to evaluate the clinical efficacy of these different IL-2 regimens in ACT with TIL, as presented in Table 1.

Toxicity

The most common toxicities during TIL therapy are due to the effects of the lymphodepleting preparative regimens and the subsequent IL-2 after TIL infusion [32]. TIL-related toxicity is less common, but patients may develop, mostly transient, dyspnea, chills and fever shortly after infusion of TIL. Other signs of toxicity develop later after infusion and may consist of melanoma associated autoimmune diseases such as vitiligo or uveitis, of which the latter promptly responds to topical corticosteroid treatment. This demonstration of autoimmune-like toxicity does not seem to be significantly correlated with response to TIL therapy [19]. In general, autoimmune-like toxicity such as uveitis, hearing loss and vitiligo after TIL therapy is much less common compared to development of these side-effects following ACT with MART-1 or gp100 specific T cell receptor (TCR) gene therapy [33]. One plausible reason for this difference could be that TIL products consist of a more polyclonal T cell population targeting more and other antigens than the homogenous T cell population in the TCR gene therapy product.

Autoimmune toxicity due to TIL therapy is not always transient, as described by Yeh et al. In this case report, a patient undergoing TIL therapy developed severe autoimmune sequelae including diffuse erythematous full-body rash, persistent panuveitis and hearing loss. The patient was treated following preparative lymphodepletion with cyclophosphamide, fludarabine and TBI 12 Gy prior to infusion of 1.4 × 10¹¹ autologous TIL and 4 doses of HD IL-2. Biopsy of the rash showed dermal CD8⁺ T cell infiltrates. Flow cytometry of ex vivo expanded T cells from biopsies of the eyes demonstrated much higher percentages MART-1 MHC multimer-positive CD8⁺ cells compared to the peripheral blood after TIL therapy. The patient showed a durable CR of the metastatic melanoma 2 years after TIL therapy [34]. Although this case report suggests a positive correlation between the occurrence of autoimmune toxicity and response to ACT with TIL, such a correlation has not yet been demonstrated in larger patient cohorts.

“Young” TILs

In most early studies, several TIL cultures were established per patient and only the tumor reactive cultures were pre-selected for further outgrowth. Tumor reactivity was detected based on IFN-γ production upon in vitro co-culturing with autologous tumor material or HLA-matched tumor cell lines [8]. In later studies, this “selected” TIL strategy was exchanged for minimally cultured “young” TILs with an initial outgrowth phase < 20 days. During “young” TIL preparation, no pre-selection on tumor reactivity is used. All TILs that are grown are used for REP, making it easier to adapt [8, 49]. Interestingly, clinical response rates with “young” TILs are comparable with “selected” TIL [35, 50], which makes “young” TIL the current standard in the field.

Besides the ease, young TILs have two other major advantages; firstly, culture time is kept to a minimum. This is important since short culture times are associated with a better clinical response to TIL therapy [35]. Secondly, this optimization step results in a higher success rate to generate a clinical product, since for some patients no autologous tumor material or matching cell line is available, or no IFN-γ production could be observed.

TIL selection

TIL products are heterogeneous products. Not only do they differ in percentage CD8⁺ versus CD4⁺ T cells, but also in tumor reactivity and antigen specificity. As discussed above, only a fraction (up to 30% in our hands) of the total population is tumor reactive. In order to enhance tumor reactivity, TIL could be enriched using a selection marker. Selecting for a tumor reactive population beforehand could ideally also shorten culture time and lower the number of infused cells.

In 2010 Rosenberg and colleagues showed that PD-1 expression is high on melanoma reactive TIL and that this marker could be used to pre-select tumor reactive cells from the bulk TIL population using FACS or magnetic bead sorting. After enrichment, the PD-1 positive T cells were expanded in standard REP protocol. Using this PD-1 selection method, in three out of five tested patients, TIL products showed enhanced tumor reactivity compared to the PD-1 negative or non-selected population [51].

In another study, Powell et al. showed that CD137/4-1BB, an activation marker for CD8⁺ T cells, could be used to select tumor reactive TILs from melanoma samples. TILs were either FACS sorted or bead selected based on CD137 expression, and also these selected cells showed enhanced tumor reactivity compared to unselected TIL. Both showed enhanced in vitro recognition of melanoma cells lines, based on IFN-γ production, and in vivo tumor control in a patient derived xenograft (PDX) mouse model was demonstrated [52]. Recently the Sheba Medical Center, Israel, demonstrated that CD137 selection could be performed with clinical grade compliant reagents. They expanded CD137 selected TILs in a large-scale manner to meet cell numbers required for patient treatment in a GMP facility. The increased anti-tumor effect was most prominent in an in vitro killing assay (using LDH release) and less prominent in IFN-γ release. Using this protocol, CD137 selected TILs were enriched for the recognition of both neo-antigens and shared antigens [45]. The Sheba Medical Center is currently running a trial using this CD137 selection strategy. Whether CD137 or PD-1 is the best marker to enrich for melanoma-reactive TIL is not known at present. Both methods will be further evaluated in clinical trials.

Our own group showed that the tumor reactivity of TIL products can be enhanced using clinical grade MHC streptamers to enrich for sub-populations of TIL with defined specificities. This strategy works for selection of TIL with both shared and neo-antigen reactivity. Importantly, the protocol can be performed under GMP conditions. A major challenge for clinical implementation of this strategy, is the requirement for knowledge of the peptide-specificity within the TIL product, before the MHC streptamers can be generated [53]. In addition, streptamers are only available for a limited number of HLA- alleles.

Several groups showed that infusion of high numbers of CD8⁺ TIL is associated with a higher objective response [17, 35]. Both total number and percentage of CD8⁺ cells is significantly correlated with objective response (p = 0.0003 and p = 0.001 respectively) [17]. In addition, the observation was made that the presence of CD4⁺FoxP3⁺ Tregs is associated with lower clinical activity of TILs [54], suggesting that CD4⁺ cells in the infusion product might negatively influence clinical activity. This hypothesis was tested in a randomized controlled trial (RCT) with TIL in melanoma patients in which CD8⁺ enriched and unselected “young” TIL were compared. This study failed to show higher clinical activity of the CD8⁺ selected TILs [46].

Genetic editing of TIL

Current rapid developments in gene editing could also further enhance TIL therapy. These developments make it technically feasible to introduce potential beneficial receptors or molecules, or the other way around, knock-down/knock-out the ones that might be reducing the effect of TIL. Rosenberg and colleagues showed that Zinc Finger nuclease can be used to down regulate PD-1 in TIL, resulting in clinical grade TIL products with an enhanced effector function and cytokine production [55]. The currently widely used CRISPR-cas9 technology is expected to further increase the possibilities for gene editing of TIL. The MD Anderson Comprehensive Cancer Center, Houston, Texas, US, uses a lentiviral vector to transduce TIL with the chemokine receptor CXCR2, which could potentially improve tumor homing [56]. This strategy is currently evaluated in the clinic (see Table 1, NCT01740557). Transient, non-viral gene delivery by mRNA could also be used as alternative for the introduction of additional chemokine receptors in TIL [57]. All these technical developments open endless potential genetic improvements of the cell products.

An overview of the current TIL production protocol and potential improvements is shown in Fig. 1.

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Fig. 1

Schematic Overview of the Current TIL Production Protocol and Potential Improvements. Currently, surgically removed melanoma metastases are processed into single cell digest or smaller tumor pieces. At this point in production, direct selection of tumor reactive cells based on activation markers such as PD-1 or CD137, or CD8⁺ T cells or multimers can be applied. TIL outgrowth currently occurs in HD IL-2. Outgrowth of TIL could be improved in the presence of alternative cytokines such as IL-7, IL-15 or IL-21 or agonistic co-stimulatory antibodies such as CD137. In addition, a variation of gene modifications of homing or co-stimulatory factors can be applied. The current REP protocol consists of addition of activating soluble anti-CD3, HD IL-2 and irradiated feeders, but may be improved by addition of alternative cytokines such as IL-7, IL-15 and IL-21 and artificial feeders may be used. Also, the current REP time may be shortened. After REP, gene modification can also be applied. The infusion procedure of TIL to the patient currently consists of a conditioning lymphodepleting regimen, usually cyclophosphamide and fludarabine and administration of HD IL-2 following TIL infusion. However, multiple studies are being conducted with adjusted doses and treatment schedules of the lymphodepleting regimen and IL-2, as are studies being conducted with TIL as combination therapy to further potentiate the anti-tumor effect of TIL

TIL as adjuvant therapy

Few studies with TIL treatment have been performed in patients with stage III melanoma. In a RCT conducted by Dreno et al., Nantes, France, 88 patients with stage III melanoma were treated with adjuvant TIL/IL-2 (n = 44) or IL-2 alone (n = 44) after surgery. Their hypothesis was that TIL treatment could be more efficacious in a setting with a minimal tumor burden. Patients receiving two infusions of 0.22–3.34 × 10¹⁰ TIL at 6 and 10 weeks post-surgery followed by daily s.c. IL-2 injection (6 × 10⁶ IU/m²) for 5 days a week for 2 weeks with each TIL infusion, showed superior relapse free survival (RFS) and OS compared to the s.c. IL-2 only [10, 59, 60]. Importantly, TIL infusions were not preceded by NMA lymphodepletion and the number of cells infused were ~ 10-fold lower compared to ‘classical’ TIL. As s.c. IL-2 is not approved as adjuvant therapy for patients with stage III melanoma, it is difficult to put the outcome of this study in perspective.

Combination therapy with TIL

Recently, results have been published from a study in metastatic melanoma patients, who were treated with the combination of a targeted agent plus TIL. In this pilot study in 11 patients with BRAF^V600E/K mutated melanoma, the BRAF inhibitor vemurafenib was given in conjunction with TIL. Patients were treated with vemurafenib for two weeks following metastectomy for the production of TIL, after which another lesion was resected. Patients were further treated according to standard protocol of lymphodepleting regimen, TIL infusion and IL-2. Vemurafenib was resumed after TIL infusion and continued for two years. Seven out of 11 patients (64%) showed an objective clinical response, two of whom had a durable response lasting up to three years [61]. These results are promising, however larger, randomized studies are needed to show the value of this approach in comparison to TIL alone. Currently, two clinical trials in which targeted therapy is being combined with TIL are actively accruing patients (NCT02354690, NCT01659151), see Table 1.

Treatment with the anti-CTLA-4 drug ipilimumab, was shown to heighten T cell infiltration into melanomas and to broaden the TIL response to these tumors [62]. In a recent clinical trial at the Moffit Cancer Center, Tampa, US, 13 patients with metastatic melanoma were treated with ipilimumab in combination with standard TIL therapy. Patients received four doses of ipilimumab (3 mg/kg), starting two weeks before metastectomy for TIL harvest, one week after resection of a metastasis, followed by two and five weeks after conditioning chemotherapy. Five out of 13 patients (38.5%) showed an OR, four of which were durable, lasting up to one year and one patient developed a CR 52 months after this treatment [11]. Response rates seen in this trial were not different from those in other TIL trials. However, these data are the first to demonstrate the feasibility if combining TIL with immune checkpoint blockade.

Currently, several trials have been initiated combining TIL with PD-1 blocking agents (NCT03374839, NCT03475134, NCT03158935, NCT02652455, NCT02621021, NCT01993719), see also Table 1. Synergism from this combination may be expected as the ex vivo grown and expanded tumor-reactive TIL are often PD-1 positive [63] and prevention of the interaction between PD-1 on T cells and PDL-1 on tumor cells by anti-PD-1 therapy around the time of TIL infusion, may render these TIL more tumoricidal.

In addition, other immunotherapy modalities such as dendritic cell vaccination and (peg-)interferon, are being evaluated in a clinical setting combined with TIL therapy. See also Table 1 for details on current recruiting trials of combinations with TIL.

TIL therapy for other solid tumor types

For decades TIL treatment has been studied in patients with mostly metastatic cutaneous melanoma. Recently, investigators were also successful in growing out tumor reactive TILs from other tumor types, such as renal cell, breast and cervical cancer. In general, the tumor reactivity of TILs from these other tumors is lower when compared to melanoma [64]. The production and reactivity of TIL products for these other solid tumor types varies, amongst others, due to the heterogeneity in mutational load, and thus neo-antigens, and lymphocytic infiltration with variations of CD4⁺ and CD8⁺ T cells [65].

Promising ORR of up to 35% have been seen in patients with metastatic uveal melanoma in an ongoing single-center, single-arm, phase II TIL study with 21 patients [66]. Despite the for this disease impressive ORR, the durability of these responses appeared short compared to what has been observed for cutaneous melanoma. A phase II trial has opened to confirm these results in a larger cohort, NCT03467516, see Table 1.

Recently, successful isolation, expansion and tumor recognition of TIL from renal cell carcinoma was reported. However, the reactivity of TIL was weaker and showed reduced functionality compared to TIL from melanomas [67]. Also in breast cancer, it is possible to isolate and expand TIL ex vivo under standard culture conditions. Four out of six randomly selected post-REP TIL samples were found to be reactive to the autologous tumor in vitro, which also showed functionality in vivo in a xenograft mouse model [12]. Recently, Stevanovic et al. demonstrated clinical responses upon TIL treatment in patients with refractory metastatic cervical cancer, with three of the nine treated patients showing objective tumor regression, two of which were durable. When possible, TILs were selected for HPV E6 and E7 reactivity, as the vast majority of cervical cancers harbor HPV oncoproteins that may act as immunotherapeutic targets for TIL [13]. Currently, a “basket” clinical phase II study is being conducted at the NIH in patients with a variety of metastatic disease, including digestive tract, breast, urothelial, ovarian and endometrial cancers, in order to provide information about rates of tumor regression when treated with TIL (NCT01174121).