Current state of using immune cells to treat diseases

In the past few decades, the potency of the immune system in the development and treatment of cancer has been a major focal point of research [1]. Although targeted therapy and immunotherapy with immune checkpoint blockade have greatly improved the survival of, amongst others, melanoma and non-small cell lung cancer patients, a large proportion of patients still develop disease progression upon these therapies [2, 3]. Adoptive cell therapy (ACT) may provide an additional treatment option for these patients and comprises the intravenous transfer of either tumor-resident or peripheral blood modified immune cells into cancer patients to mediate an anti-tumor function. Currently, ACT can be classified into three different types with each their own mechanism of action, namely ACT with tumor-infiltrating lymphocytes (TIL), ACT using T cell receptor (TCR) gene therapy, and ACT with chimeric antigen receptor (CAR) modified T cells [4]. The use of other immune cell types such as natural killer cells as a basis for cell therapy is also an area of current research. However, many hurdles have to be overcome in order for this to be an effective anti-cancer treatment [5] and lie beyond the scope of this review.

The first studies with TIL were performed by Rosenberg and coworkers at the Surgery Branch in the National Institutes of Health (SB, NIH, Bethesda, Maryland, US), where TIL were grown from different murine tumors and showed anti-tumor activity in vivo [6]. The current TIL therapy consists of ex vivo expansion of TIL from resected tumor material and adoptive transfer into the patient following a lymphodepleting preparative regimen and subsequent support of interleukin-2 (IL-2). With this regimen, remarkable objective tumor responses of around 50% have been achieved in patients with metastatic melanoma in several phase I/II clinical trials [7–9]. After the successes seen with TIL in melanoma patients, the production of TIL from other solid tumor types has also been studied. Up until now, it has been possible to grow out TILs from non-melanoma tumor types such cervical cancer [10], renal cell cancer [11], breast cancer [12], and non-small cell lung cancer [13] with varying rates of tumor reactivity.

Next to the naturally occurring TILs in tumors and the thereupon-based treatment options, peripheral blood T cells can be isolated and genetically modified in vitro to express TCRs that target specific tumor antigens for the use of ACT. With the use of this method, large pools of tumor specific T cells can be generated [14, 15], with potent anti-tumor activity and objective clinical responses observed in up to 30% of treated patients [16]. For the recognition by the modified TCR, antigen presentation via the major histocompatibility complex (MHC) is required. However, it is well known that many cancer types can escape T cell-mediated immune responses by downregulation or loss of their MHC expression [17]. To circumvent the need for the presence of MHC on tumor cells for the recognition by tumor-specific T cells, artificial receptors such as CAR molecules have been developed. ACT with CAR-modified T cells holds the capacity of the same effector function as TCR-modified T cells, but independently of MHC expression [18]. Besides the use of protein antigens, other antigens such as carbohydrates [19] or glycolipid antigens [20, 21] have also been explored. Impressive clinical responses have already been seen in hematological malignancies with CD19-specific CAR T-cells [22], which led to the exploration of using CAR therapy in solid tumors as well [23]. Table 1 summarizes these three different treatment modalities of ACT and Fig. 1 shows an overview of the process for adoptive cell therapy with TIL, TCR gene therapy and CAR-modified T cells.

Although most studies with ACT in solid tumors have been performed in melanoma, the role of ACT in the treatment of other tumor types is growing. Recently, an overview of initiated trials conducted with ACT since May 2015 was published by Fournier et al. [27], where an impressive 121 new clinical trials were described (including ACT in non-solid tumors). This illustrates the need for up-to-date knowledge on ACT in this quickly developing field. The aim of this review is to give a comprehensive overview of the previous developments and the current status of ACT, as the potential of ACT as treatment modality in cancer continues to rise.

The presence of TIL in neoplastic tissue is thought to indicate an anti-tumor immune response by the host and correlates with clinical outcome in several tumor types, especially in melanoma [28, 29]. Dr. S. Rosenberg (SB, NIH, Bethesda, Maryland, US) was the first to demonstrate the anti-tumor activity of TIL in vivo in murine models in the 1980s of the past century [6]. Combining T cell growth factor IL-2 with the TIL infusion product resulted in a greater therapeutic potency of TIL compared to lymphokine-activated killer (LAK) cells produced from peripheral blood lymphocytes in the presence of IL-2 in mice with metastases from various tumor types. Addition of cyclophosphamide to TIL and IL-2 further potentiated the anti-tumor effect of TIL [30]. These early murine studies formed the basis for the original and still most commonly used TIL treatment protocol.

In the original treatment protocol of TIL in metastatic melanoma, patients underwent resection of one or more metastases with a total diameter of at least 2–3 cm. The resected tumor was fragmented or enzymatically digested and subsequently cultured in the presence of IL-2, which resulted in proliferation of TIL. This initial outgrowth phase took approximately 14 days. Once culture consisted mostly of CD3⁺ T cells, their specificity was tested during a short culture in the presence of an autologous or HLA-matched tumor cell line by quantification of interferon-γ (IFN-γ) [7]. This selection step, however, was time-consuming and complex. Follow-up studies showed that TIL production without this pre-selection for tumor reactivity, so-called “young TIL,” resulted in comparable clinical responses [31, 32] and became the current standard treatment protocol. At least 50 × 10⁶ TILs from this initial outgrowth phase are required to be further expanded in a rapid expansion protocol (REP) in the presence of a soluble anti-CD3 antibody, IL-2 and irradiated allogeneic or autologous feeder cells. During this 14 days lasting expansion phase, up to approximately 1 × 10¹¹ cells are obtained. These TILs are harvested and prepared for infusion into the patient [33]. Prior to infusion, patients will undergo lymphodepleting, but non-myeloablative (NMA) chemotherapy consisting of 2 days intravenous (i.v.) cyclophosphamide (60 mg/kg) followed by 5 days fludarabine (25 mg/m²). Shortly after infusion of TIL, patients receive i.v. high-dose (HD) bolus IL-2 (720,000 IU/kg every 8 h until maximal tolerance) [7, 24, 34]. Subsequent support with IL-2 is thought to further enhance the survival and clinical efficacy of TIL [35].

Multiple studies have been performed evaluating the role of preconditioning lymphodepleting regimens. Lymphodepleting regimens cause a short, but deep lymphopenia and neutropenia, with full bone marrow recovery within 7–10 days, not requiring hematopoietic stem cell support [36]. Murine models had shown that response rates upon TIL improved after prior lymphodepletion by total body irradiation (TBI) or chemotherapy [30, 37]. These models showed that depletion of endogenous lymphocytes created “physical space,” resulted in less competition for homeostatic cytokines IL-7 and IL-15 [38] and removed immunosuppressive lymphoid and myeloid populations [39]. Incorporating an intensified lymphodepleting regimen by combining cyclophosphamide/fludarabine with either 2 Gy or 12 Gy TBI, improved the overall response rate from 49% to respectively 52% and 72% in small cohorts of 25 metastatic melanoma patients. Significantly higher levels of IL-7 and IL-15 were measured in patients treated with the intensified lymphodepleting regimen (p = 0.02 and p = 0.005 respectively). However, this intensified regimen did result in more acute toxicity and prolonged organ dysfunction and required hematopoietic stem cell support for bone marrow recovery [34]. In a randomized controlled phase II trial by the NIH in 2016, the clinical benefit of addition of 12 Gy TBI to NMA chemotherapy could not be reproduced [40]. A currently recruiting phase II trial conducted by the Sheba Medical Center, Israel, evaluating the effect of a reduced intensity lymphodepleting regimen prior to TIL is expected to give further insight into the optimal preparative regimen (NCT03166397↗).

Most studies conducted with TIL have been performed in patients with advanced (cutaneous) melanoma, but the place of TIL in the current standard treatment protocol is still being investigated. Promising clinical responses have been observed in patients with metastatic uveal melanoma [41] and may provide a novel treatment modality for this group of patients, as no standard treatment is available yet. The currently recruiting phase III randomized clinical trial comparing ipilimumab to TIL treatment (NCT02278887↗) should provide more evidence for the role of TIL as anti-tumor treatment in metastatic melanoma. Successful production of TIL was also achieved in renal cell carcinoma [11], breast cancer [12], cervical cancer [10], gastrointestinal cancers [42, 43], cholangiocarcinoma [44], pancreatic cancer [45], head and neck cancer [46], ovarian cancer [47], and non-small cell lung cancer [13]. However, the antitumor reactivity of TIL obtained from these other solid tumors still remains a challenge. It has been demonstrated that TILs isolated from melanoma show consistent antitumor reactivity [48] and that high mutational load and high neo-antigen rates are significantly associated with clinical benefit upon TIL therapy in patients with metastatic melanoma [49]. The production and reactivity of TIL products from other solid tumor types is highly variable, most likely as a result of heterogeneity in mutational and neo-antigen burden, in lymphocytic infiltration with variations of CD4⁺ and CD8⁺ T cells, in myeloid infiltrate composition and other as yet unknown factors [50]. Further research is needed in this field in order to potentiate TIL treatment for other solid tumors.

Antigen receptor gene engineering with TCRs or CARs is a developing and promising new anti-tumor modality [15]. In contrast to TIL, peripheral blood lymphocytes are enhanced to create anti-tumor reactivity by transduction with tumor-specific receptors. This modification can be performed with relevant tumor-reactive TCR for TCR gene therapy or synthetic antibody-based receptors for CAR T cell therapy [36].

The toxicity observed with ACT can grossly be divided in three groups: toxicity due to the lymphodepleting preparative regimen, immune-related toxicity and cytokine-related toxicity. During treatment with TIL, toxicities are predominantly caused by the lymphodepleting preparative regimens, resulting in pancytopenia and febrile neutropenia, and the supportive IL-2 infusions [8, 107, 108]. These toxicities are also seen in TCR gene therapy [64] and CAR therapy [97]. The most prevalent side effects seen in TIL treatment due to IL-2 include chills, high fever, hypotension, oliguria, and edema due to the systemic inflammatory and capillary leak syndrome effects and can usually be treated with supportive measures [109]. Reports have been published of autoimmune phenomena such as vitiligo or uveitis, however, these side effects are not frequently seen and uveitis usually responds well to local corticoid treatment [108].

The potency of TCR therapy lies with the recognition of antigens on tumor cells, often not tumor-specific and thus also expressed on healthy tissues. This can lead to the occurrence of “on-target, off-tumor” toxicity [36]. Targeting melanoma differentiation antigens such as MART-1 and gp100 can result in severe skin rash, uveitis, and ototoxicity due to expression of these antigens in these organs [16] and these effects seem to be dose-dependent [110]. Some of the used TCR, such as NY-ESO-1 and MAGE-A3 have undergone affinity maturation in conducted clinical trials to increase the affinity of the TCR for the target MHC-peptide complex [64, 66]. This process may increase the chance of cross-reactivity to other targets as well, and may have caused some of the severe toxicities that were seen thus far with TCRs directed at C/T antigens. The C/T antigen MAGE-A3 is not known to be present in normal tissue, but two separate TCR-based therapies directed to MAGE-A3 did result in fatal neurotoxicity and cardiotoxicity. Neurological complications may be due to cross-reactivity to MAGE-A12 which is expressed in a subset of neurons in the brain and the cardiac events may be due to cross-recognition of the muscle protein Titin [66, 111], perhaps as a result of TCR affinity enhancement. On the other hand, treatment with the C/T antigen NY-ESO-1 specific TCR transduced T cells so far seem to be safe [64]. B cell aplasia is an expected “on target off tumor” side effect of CD19 CAR T cell therapy and can be managed with replacement therapy with intravenous immunoglobulin [79].

Another common toxicity seen in ACT is cytokine release syndrome (CRS). CRS is a non-antigen-specific adverse event and is a result of activation most probably of the infused T cells through antigen recognition, leading to massive cytokine release, including IFN-γ, IL-1, and IL-6 [112]. Toxicity from CRS has a very heterogeneous presentation, but usually involves fever, hypotension, tachycardia, and respiratory insufficiency and it is potentially fatal, as described by van den Berg et al. in a case report of a patient with metastatic melanoma treated with a TCR recognizing MART-1 [110]. The severity of CRS is correlated with tumor burden [26, 113]. Severe and life-threatening CRS can effectively be treated with tocilizumab (human monoclonal antibody against the IL-6 receptor) or siltuximab (monoclonal antibody against IL-6) and sometimes corticosteroids [112, 114, 115]. Additional targeted immunosuppressive agents like infliximab (anti-TNF-α antibody) and anakinra (anti-IL-1R antibody) have been used in some very severe cases [112], the latter of which has recently also been shown to be effective against neurotoxicity in CAR T cell therapy in a murine model [116].

Neurological complications, including confusion, delirium and occasional seizures and cerebral edema, occasionally resulting in death, have been observed with the use of CD19-specific CAR T cells [117]. The underlying pathogenesis of this neurotoxicity remains currently unknown. Recently, endothelial cell activation and increased blood-brain barrier permeability were found to play a role at the initiation of this toxicity [118]. Intensive research is currently ongoing to further elucidate the underlying pathogenesis of both CRS and the neurotoxicity, identify useful biomarkers and optimize current treatment algorithms [119].

Lastly, an IgE-mediated anaphylactic reaction has been observed in a patient treated with autologous T cells electroporated with mRNA encoding for a CAR derived from a murine antibody to mesothelin [120]. This reaction may have been induced by “foreign” CAR moieties and current strategies for CAR therapy involve the use of humanized or even fully human scFv to circumvent these IgE-mediated responses.

The ultimate goal of adoptive cell therapy for malignancies is to create an optimized personalized cellular product solely reactive to the tumor. In the past decades, the production of TIL, TCRs, and CARs have all undergone developments to improve its efficacy as anti-cancer treatment. TIL are a heterogeneous cell product and the presence of antigen-reactive TIL is a key determinant of anti-tumor reactivity [121]. Enrichment for tumor-reactive TIL through CD137 [122] or PD-1 [123] selection is currently being investigated as a method to increase the anti-tumor reactivity.

Genetic editing of TIL may also improve its functionality, as has already been demonstrated with Zinc finger nuclease engineering which decreases the PD-1 expression in TIL, improving the efficacy [124].

In TCR therapy, it is of major importance that targetable antigens are identified which are expressed on the tumor, but not on healthy tissue to decrease the “on-target, off-tumor” toxicity and to further potentiate its effector function. Currently, oncogenes such as BRAF^V600E driver mutations are also being explored as possible therapeutic targets for ACT and have already shown clinical activity in a patient with advanced melanoma [125]. HPV antigens E6 and E7 are being explored as targets for ACT as treatment of HPV-associated cancers [10, 69]. Neo-antigens that arise as a result of tumor-specific mutations appear exquisite targets for TCR gene therapy as these antigens are, like viral antigens, fully foreign to the immune system. Gene therapy targeting these antigens are also being developed.

As in ACT with TCR gene therapy, the main challenges in CAR therapy comprise new target discovery, reduction of toxicity, and improvement of cell trafficking. Because of the correlation between tumor burden and toxicity from CAR T cell therapy [26, 113], tumor reduction before therapy or dose adaptation strategies could possibly be applied to reduce side effects. More promising are current investigations with CAR T cells engineered to contain suicide genes or switches (for example iCasp9) which are evaluated in preclinical and clinical studies [126]. CAR T cells seem to express inhibitory receptors such as PD-1 in an exhausted state, which upregulates PD-L1 on tumor cells. Combinatorial approaches of CAR T cell therapy and PD-1 blockade have resulted in improved CAR T cell activity and tumor eradication in preclinical experiments and clinical studies are currently ongoing [127, 128]. Furthermore, the combination of CAR T cell therapy with oncolytic virus-driven production of a bispecific T cell engager showed enhanced efficacy in a mouse model compared to both monotherapies [129].

In conclusion, ACT represents a personalized immunotherapeutic approach that has developed rapidly in recent years. Great successes have already been seen with the use of TIL treatment in melanoma and CAR therapy in hematologic malignancies. However, further optimization of this promising treatment modality is warranted to enhance the anti-tumor effect and reduce the associated toxicity. More than 100 clinical trials have been initiated since 2015 and are currently being conducted with ACT [27] and should provide new insights in the efficacy and further developments of this treatment modality.

All authors contributed equally to writing the manuscript. All authors read and approved the final manuscript.

Not applicable. All cited published original research in this manuscript are publically available.

Maartje W. Rohaan, Email: m.rohaan@nki.nl

Sofie Wilgenhof, Email: s.wilgenhof@nki.nl.

John B. A. G. Haanen, Email: j.haanen@nki.nl