Efficacy of Anti-Cancer Immune Responses Elicited Using Tumor-Targeted IL-2 Cytokine and Its Derivatives in Combined Preclinical Therapies

1. Introduction

Cancer therapies have been revolutionized by the use of immune checkpoint inhibitors (ICI) both as alone treatments and in combination with other approaches [1,2,3,4,5,6,7,8,9,10]. Initially applied for patients with metastatic melanoma and metastatic renal cell carcinoma, these therapies have since been extended to other cancer types, achieving excellent results and, in some cases, definitive cures [1,4,8]. However, it became apparent that a significant portion of the patients did not respond to ICI [11,12,13,14]. Among the various potential reasons for this, two critical factors must be considered: the inhibitory role of the TME on immune cells and its proactive role in promoting carcinogenesis and tumor progression [15,16,17,18,19,20].

Of note in 2011, Hanahan and Weinberg [21] redefined and highlighted several new key hallmarks of cancer, including enabling characteristics that were initially postulated in 2000 [22]. Of particular importance are three hallmarks that play fundamental roles in tumor dynamics: “evading immune destruction”, “tumor-promoting inflammation”, and “immune orchestration of angiogenesis”. These characteristics underscore the essential and dual role of the immune system in neoplastic diseases.

Immune cells, particularly innate immune cells such as macrophages and natural killer (NK) cells, can exhibit potent anti-tumor activities, including phagocytosis and cytotoxic functions. [23,24,25]. However, at the same time, macrophages and NK cells upon interactions with the tumor and TME, can become protumor effectors with low cytotoxic capacities and display immunosuppressive and pro-angiogenic behaviors [26,27,28].

In this context, Hanahan recently suggested new emerging characteristics of tumor cells: “unlocking phenotypic plasticity,” “non-mutational epigenetic reprogramming,” “polymorphic microbiomes,” and “senescent cells”, making the study of malignant tumor growth even more complex and intricate [29]. However, increasing experimental data have begun to clarify the protumor or antitumor roles played by different types of immune or stromal cells, in particularly in promoting or suppressing tumor growth, reinforcing or inhibiting metastasis and contributing to therapy.

Hanahan defined “unlocking phenotypic plasticity” as a potential new hallmark of cancer, referring specifically to malignant cells. However, from the point of view of the immune cells in the TME, it appears that these cells can also exhibit high plasticity in both their phenotype and function. Indeed, as a result of tumor signaling and the release of soluble factors or extracellular vesicles, immune cells can become highly plastic, altering their phenotype and function. These alterations, induced by interactions with cancer, stromal and endothelial cells, as well as cytokines/chemokines in the TME, can lead to impairment of cytotoxic activities, the development of immunosuppressive features and, in many cases, the acquisition of pro-angiogenic activities.

Among these factors, cytokines play a crucial role in shaping the cancer-induced immune cycle by prompting various processes such as tumor antigen expression, antigen presentation, priming and activation of immune cells, recruitment of effector immune cells to the tumor site, and ultimately facilitating cancer cells elimination within the TME [30]. On the other hand, proinflammatory cytokines might have both protumor or antitumor effects. For IL-2, several lines of evidence have shown that this cytokine serves as a crucial regulator for the activation of helper/regulatory T cells, cytotoxic T cells and NK cells, influencing their proliferation, differentiation, and contributing to both pro- and anti-inflammatory immune responses. Some specific cytokines are identified as promising cancer immunotherapies. They function by adjusting the immune response against cancer cells and by directly displaying anti-cancer effects, including inhibiting proliferation and promoting apoptosis. Cytokines have a substantial track record as anticancer treatments, starting from the 1970s, when interferon (IFN)-α and IL-2 were the first cytokines utilized in cancer therapy [30]. Among the various types of immunotherapies, cytokine therapy, shows high promise in cancer treatment. IL-2 treatment has shown significant efficacy in metastatic melanoma, renal cancer, and advanced non-Hodgkin’s lymphoma. However, following these early advancements, the clinical translation of these molecules has been significantly restricted due to their broad and diverse biological effects across various cell types. These characteristics, combined with suboptimal pharmacokinetics, such as short half-lives, have impeded the effective systemic administration of cytokines, primarily due to severe dose-limiting toxicities [31]. In fact, severe adverse reactions, such as vascular leakage syndrome, underscore the need for careful administration under the supervision of an oncologist in a hospital setting. To address these challenges, novel engineering strategies been developed to broaden the therapeutic range, extend the duration of pharmacokinetic effects, improve tumor-specific targeting, and minimize adverse effects, thereby enhancing the overall efficacy of the therapy [31]. Herein, we focus on discussing the impact and relevance of anti-cancer immune effectors induced by IL-2 immunotoxins (IT), IL-2 immunocytokines and IL-2 derivatives or variants in different combined therapies.

2. Main Features of IL-2 and Tumor Specific Targeting of IL-2

Since its discovery in 1976 and the subsequent understanding of its vital role as a lymphocyte T-cell growth factor, the cytokine IL-2 was approved in the 1990s for use in patients with advanced stage cancer, including renal cell carcinoma and later for metastatic melanoma. IL-2 is a 15.5 kDa glycoprotein involved in immune response [32,33,34]. IL-2 exerts its function by interacting with IL-2 receptors (IL-2R) [34,35,36]. The IL-2R is comprised of three different subunits: IL2Rα (CD25), IL2-Rβ (CD122) and IL-2Rγ (CD132). The strongest binding affinity is observed when IL-2 binds to the trimeric IL-2Rαβγ complex, though it can also bind to the dimeric IL-2Rβγ [37,38]. This implies that the lymphocytes expressing the trimeric form of the IL-2R will respond promptly to 10-100 folds lower amount of IL-2 than cell population expressing the dimeric βγ IL-2R. Indeed, regulatory CD4⁺ T cells and activated T cells display high CD25 expression and respond to IL2 very efficiently [39,40]. On the other hand, CD8⁺ memory T cells and NK cells, which express IL-2Rβγ⁺, require higher doses of IL-2 than regulatory T cells. Upon binding to IL-2R, the signal transduction cascade is initiated through the heterodimerization of the β and γ subunits, leading to the activation of Janus kinases (JAK1 and JAK3) and the subsequent phosphorylation of the β chain. This activation recruits and phosphorylates STAT transcription factors, primarily STAT5, which dimerize, translocate to the nucleus, and bind to DNA to promote the transcription of genes responsible for T and NK cell activation and proliferation. This signaling pathway also engages downstream PI3K/AKT/mTOR and MAPK/ERK pathways [37,38,41,42].

High-dose IL-2 regimens have shown considerable efficacy, inducing potent cytolytic responses and expansion of CD8⁺ T and NK cells in approximately 10% of patients. However, a substantial portion of patients experienced severe adverse effects. Among these, is vascular leak syndrome (VLS) and severe pulmonary edema, attributed to direct endothelial cell action inducing vasodilation, as well as fever, hypotension, cytopenia and organ dysfunction [43,44,45,46,47]. Both innate and adaptive immunity influence VLS onset, with NK cells contributing and Treg cells mitigating severity [48,49,50,51]. While systemic IL-2 administration for cancer has resulted in toxicity, local application, particularly intratumoral administration, shows promise [52,53,54]. However, it is evident that IL-2 can play a dual role in anti-tumor immunity. On one hand, it can trigger the generation of anti-tumor cytotoxic effector cells, such as CD8⁺ T cell antigen specific lymphocytes and NK cells, on the other hand, it can stimulate the immunomodulatory function of Treg cells, leading to the inhibition and exhaustion of the anti-tumor immune response. Notably, the use of low doses of IL-2 preferentially activates Treg cells, resulting in the inhibition and weakening of anti-cancer cytolytic responses [55,56,57,58,59].

Additionally, the half-life and the efficacy of IL-2 should be enhanced. It is crucial that these modified properties of IL-2 do not lead to increased vascular toxicity or heightened immunoregulatory effects [60,61]. The features of chimeric cytokines such as IL-2 and IL-15 have been extensively described in recent studies [62]. Indeed, the generation of several mutated IL-2 as well as the pegylation or production of IL-2 prodrugs can increase the half-life and the efficacy of IL-2 as detailed summarized [62]. The primary challenge to address is targeting IL-2 to the tumor to prolong its effect at this specific site; this to limit IL-2 interaction with the α-chain of IL-2R on endothelial and pulmonary cells which can lead to severe, life-threatening effects [63,64,65,66]. The solution of this issue lies in targeting the tumor with IL-2 in association with of an anti-tumor specific antibody generating an immunocytokine.

A detailed list of immunocytokines is reported by Ren et al. [67,68,69,70,71,72,73] and some of these immunocytokines will be discussed later on in this review. It is important to note that the antibody can be directed either to tumor cells or to components of the TME, such as mesenchymal stromal cells or tumor-associated fibroblasts. This targeting could enhance the local immune response by activating effector cells, such as CD8⁺ T cells and NK cells. Additionally, IL-2 can be part of a fused IT, as described in the next paragraph [62,74,75].

3. IL-2 Immunotoxins as Antibody-Drug Conjugates to Target and Fight Cancer Cells

The targeting of a tumor with a cytotoxic drug is one of the easiest ways to kill proliferating tumor cells [76,77,78]. This targeting is more specific when the cytotoxic drug is linked to a molecule able to recognize mainly the tumor cell [79]. This is achieved using antibody-drug conjugates (ADC) or proteins generated by molecular engineering, which are composed of a toxin and an antibody fragment capable of interacting with a tumor- specific or tumor-associated antigen [80,81,82,83,84]. In this way, the protein carrying the toxin can enter into the tumor cell, allowing the toxin to specifically kill the cell [85,86,87]. For example, it has been shown that a fusion protein, composed of the truncated diphtheria toxin, IL-2 and an anti-CCR4 antibody generating a bispecific IL2-CCR4 IT, can be used for the treatment of cutaneous T-cell lymphoma (CTCL) [88]. CTCL can indeed express good levels of IL-2Rα and CCR4 antigens, making these two receptors the optimal targets for delivering this IT. Importantly, this complex has demonstrated significant efficacy in prolonging the survival of mice bearing a CTCL tumors. Notably, it was found that this IT was more effective than the anti-CD30 ADC, Brentuximab-Vedotin. Interestingly, the combo of this ADC with the IT has been shown to produce a stronger anti-CTCL response than using either the ADC or the IT alone (Figure 1).

This indicates that the combination of these two drugs can enhance the therapeutic response in this murine model [89]. It is of note, that the IT can target not only CTCL cells but also the infiltrating Treg cells decreasing their immunosuppressive effects. This is an example of how an IL-2 IT can influence the anti-tumor response. Some recent data indicated that the CCR4-IL2 bispecific IT has a favorable safety profile in rats and minipigs animal models, suggesting its possible future use in human clinical trials [90].

However, it is important to consider that IL-2 based IT may also interact with anti-tumor effector cells. In this context, it has been shown that Denileukin Difititox (DD), which is composed of IL-2 fused to diphtheria toxin can enhance the anti-tumor immunity by interacting with the IL-2Rαβγ trimeric form selectively expressed on Treg cells, leading to their depletion [91,92]. These findings have been challenged by some observations indicating that DD administration in cynomolgus monkeys can lead to significant and long-lasting depletion of peripheral blood CD16⁺CD8⁺NKG2A⁺CD3^- NK cells, beside a strong but transient elimination of peripheral Treg cells [93]. However, this depletion can be considered an unwanted side effect, as activated NK cells are important anti-tumor effectors. Interestingly, co-administration of DD and IL-15 has been shown to prevent DD-induced NK cell depletion both in vitro and in vivo, without affecting the strong depletion of Treg cells. This protective effect is attributed to the interaction of IL-15 with IL-2Rβγ on NK cells [91,92]. Taken together, these findings suggest that the use of IL-2-based IT should be thoroughly analyzed, with consideration given to the potential use of other factors, such as IL-15, to modulate their effects.

4. Immunotherapy Using Combined Tumor Microenvironment-Targeted IL-2 Cytokine

It quickly became evident that new biotechnological tools were needed for the clinical application of IL-2 to overcome important negative factors in cancer therapy. As a result, various IL-2 immunocytokines, which consist of IL-2 fused to antibodies targeting different tumor or TME antigens, have been developed and evaluated in tumor mouse models, yielding encouraging results [93,94,95,96,97,98,99,100,101,102,103]. Antibody-cytokine protein fusion combinations show promise in enhancing efficacy, when used in combination with other treatments at the appropriate time. While pro-inflammatory immunocytokines, including IL-2, can trigger side effects, these are generally of low intensity and can be managed through careful dosing and infusion strategies. Various cytokines such as TNF-α, IL-12, and IL-2 have been employed as they can stimulate different immune cell subsets [104,105,106,107,108,109,110]. By fusing IL-2 with antibodies targeting tumor-associated antigens, researchers aimed to localize cytokine activity to the TME, employing diverse targeting strategies and modulating pharmacokinetic properties with different antibody formats [111]. Clinical studies using IL-2 immunocytokines, particularly in combination setting with other biopharmaceuticals, have demonstrated efficacy in inducing anti-tumor immune responses mediated by CD8⁺ T cells, NK cells, and macrophages (https://www.clinicaltrials.gov), as recently reviewed by Raeber et al. [112].

Diverse tumor or tumor stroma antigens has been targeted with IL-2 immunocytokine including carcinoembryonic antigen (CEA), CD20, CD30, disialoganglioside 2 (GD2), epithelial cell adhesion molecule (EpCAM), fibroblast activation protein α(FAP), extracellular matrix (ECM) proteins preferentially expressed in the tumor vasculature, such as extra domain A of fibronectin (A-FN), extra domain B of fibronectin (B-FN), and tenascin-C. The selection of ECM tumor proteins is based on the fact that some isoforms are highly expressed in the TME while they are either absent or weakly expressed in healthy tissues [113,114]. An alternative approach to using IL-2 TME specific therapy is the targeting of IL-2 to CTLs and NK cells via NKG2D surface molecule. For this purpose, a mutant moiety of IL-2, which lacks the binding capacity to CD25, was developed. This construct is formed by using a virally high-affinity encoded NKG2D ligand, known as orthopoxvirus major histocompatibility complex class I like protein (OMCP) [115]. It was shown that OMCP-mutIL-2 in a Lewis lung carcinoma mouse model was effective in inducing a strong NK cell-mediated therapeutic responses.

One of the most studied IL-2 immunocytokines has been L19-IL-2, targeting the neoangiogenesis tumor-specific B-FN isoform. L19-IL-2 has been evaluated in various tumor contexts in mouse models, where it has shown promising results in combination with other anti-tumor therapies. For example, when combined with anti-CTLA-4 ICIs or another immunocytokine consisting of L19 fused with TNF-α, it led to complete tumor eradication in teratocarcinoma and colon carcinoma mouse models [98]. Similarly, complete remission was achieved in the treatment of B-cell lymphoma in a xenograft mouse model with L19-IL-2 in combination with rituximab (anti-CD20 monoclonal antibody, mAb) [96]. Additionally, Cazzamalli et al. reported interesting preclinical data showing that L19-IL-2, in combination with a small molecule-drug conjugate selectively targeting cancer cells expressing carbonic anhydrase IX, exhibited high specificity and efficacy [116].

Other interesting preclinical results involving L19-IL-2 demonstrated its cooperation with various chemotherapeutics, as well as its effectiveness when intratumorally injected in melanoma and sarcoma mouse models [99]. Building on these promising findings, a phase II clinical trial was initiated for advanced melanoma patients, testing a single injection of L19-IL-2 either alone or in combination with L19-TNF [117].

Concerning IL-2 immunocytokines combined with anti-angiogenic treatments, the critical role of the cell surface heparan sulfate proteoglycan syndecan-1 (SDC1, CD138) in neovascularization, vasculogenic mimicry (VM), and tumor progression is well established. Its ectodomain could be made soluble through the action of the ADAM17 enzyme, and it acts as a coreceptor for several angiogenic molecules (VEGF, FGF-2 and others) or it could be physically associated with VEGFR-2 [118,119]. In xenograft melanoma model, it has been demonstrated that the combination of L19-IL-2 with anti-SDC1 OC-46F2 mAb resulted in complete inhibition of melanoma growth for up to three months after tumor injection in 71% of the treated mice [96]. More recently, in an ovarian carcinoma model, it has been confirmed that SDC1 and tumor angiogenic B-FN isoform play pivotal roles in VM and that combined treatment using L19-IL2 and an anti-SDC1 46F2SIP antibody was effective in reducing the expression of EMT markers and loss of cancer stemness traits, which were correlated with the inhibition of VM in mice [119].

However, several research groups have made significant efforts to obtain compounds with modified IL-2 binding capacity, i.e. specifically by designing sequences that retain binding to the IL-2Rβ and IL-2Rγ (CD122 and CD132, respectively), while abolishing binding to IL-2Rα (CD25), with the goal of minimizing toxicity. Levin and colleagues developed an engineered IL-2, referred to as a “superkine”, which has an enhanced binding affinity for the IL-2βR [120], leading a superior induction of CTL with minimal activation of Treg cells. Similarly, Sun et al. constructed an IL-2 immunocytokine comprising a tumor-targeting antibody with a super mutant IL-2 (sumIL-2), which has enhanced binding to CD122 and weak binding to CD25 [121]. The sumIL-2 is incorporated in place of one Fab of an anti-EGFR mAb, resulting in the construct Erb-sumIL2. Importantly, the combination of PD-L1 ICI with sumIL-2 therapy has shown a synergistic effect in controlling advanced tumors.

Another different strategy was developed by the Doberstein team [122]. Indeed, it has been made an IL-2 with high binding affinity for CD122 and low binding affinity for CD25 conserving the same amino acid sequence of wild-type (WT) IL-2. This IL-2 has been conjugated to multiple releasable polyethylene glycol (PEG) chains (a total of six chains), which effectively rendered this new prodrug IL-2 an IL-2Rβ agonist. This design, named Bempegaldesleukin (or NKTR-214), leverages the molecular location of the PEG molecules to achieve selective stimulation of CD122, enhancing its therapeutic potential [122].

As of 2023, no IL-2 enhanced compounds had been approved for the treatment of cancer patients, with the exception of NKTR-214. NKTR-214 completed phase 3 studies but failed to meet its primary endpoints in both metastatic melanoma (PIVOT IO-001 trial) and advanced renal cell carcinoma (PIVOT-09) [111].

Klein and colleagues developed an IL-2 variant (IL-2v) immunocytokine that lacks IL-2Rα binding capacity. This IL-2v is fused with a mAb that is specific to CEA or FAP, and it contains an inert Fc domain that does not have the ability to activate FcγR-bearing cells [101,123]. The IL2v part is derived through structure-based mutation of key residues in the CD25 subunit of IL2R to prevent binding to IL-2Rα while preserving its affinity for IL-2R. Notably, both compounds used in combination therapy demonstrated superior activity compared to conventional IL-2-based immunocytokines. These engineered compounds were able to trigger and reinforce NK cell and CD8⁺ effector T cell activation via IL-2Rβγ both in the periphery and within the TME. Furthermore, they synergized with anti-PD-L1 ICI therapy, as well as with trastuzumab and cetuximab chemotherapies, enhancing the overall anti-tumor response [101,123].

The in vivo anti-cancer immune response of FAP-IL2v in various combination immunotherapies was investigated in both xenograft and syngeneic murine tumor models. FAP is low expressed or absent in healthy adult tissues but is highly expressed on cancer-associated fibroblasts and pericytes in the majority of human epithelial cancers.

When combined with different therapeutic antibodies able to trigger antibody dependent cellulare cytotoxicity (ADCC), such as the anti-EGFR antibody cetuximab, FAP-IL2v significantly extended the median survival compared to single-agent treatments of BALB-neuT genetically engineered mice that spontaneously developed breast tumors. Notably, mice that responded to this therapy showed increased tumor infiltration by CD3⁺ T cells, NK cells, and CD68⁺ macrophages, highlighting the enhanced immune response facilitated by the combination therapy [123].

5. Therapeutic Relevance of Enhancing Effector Cytolytic CD8⁺ T Cell Responses Induced by the Combination of Immunocytokine IL-2 with PD-1 Cis-Targeting

A key objective in cancer immunotherapy is the activation of non-exhausted, functional cytolytic CD8+ T effector responses, alongside the activation of NK effector cells. T cell exhaustion is often associated with the expression of markers such as PD-1, TIM-3, and CD39, which limit effective anti-tumor immunity [124,125,126,127]. A promising therapeutic strategy to overcome T cell exhaustion was demonstrated with the experiments reported by Klein’s group [128]. Indeed, it has been developed an IL-2v immunocytokine targeting PD-1 in cis (named PD1-IL-2v) and directed to the FAP tumor stroma antigen [128]. The IL-2v variant avoids binding to CD25, thus limiting Treg cell expansion and reducing the immunosuppressive effects commonly observed with WT IL-2 therapy. Importantly, this modification enhanced the functional activation of the stem-like CD8⁺ T cells (TCF1⁺ PD-1⁺), a subset critical for durable immune responses. This effect is mediated by strong STAT5 phosphorylation, which boosts the activation of these T cells.

Similarly, the murine PD-1-IL-2v (muPD-1-IL-2v) also induced more potent CD8⁺ T cell effectors in the C57/BL6 model in syngeneic pancreatic ductal adenocarcinoma model Panc02 [128]. This CD8⁺ T cell effector population can induce GM-CSF production, as well as higher concentration of granzyme B. It is notable that GM-CSF is a cytokine involved in dendritic cell activation and in potentiating T cell cytotoxic effector functions [129]. Interestingly, the highest therapeutic efficacy of muPD-1-IL-2v was further demonstrated in several in vivo mouse models, such as MCA205 tumor, B16-F10-OVA and RipTag5 pancreatic neuroendocrine tumors refractory to ICI [130]. Furthermore, Tichet et al. showed that muPD1-IL-2v was able to induce remarkable tumor regression and increased survival in combination with anti-PD-L1 [128]. Notably, this therapeutic combo generated and expanded polyfunctional effector memory CD8⁺ TILs with significantly higher levels of granzyme B, IFNγ, and TNFα compared to than CD8⁺ TILs isolated from mice treated with only muPD1-IL2v. This IL-2v immunocytokine entered clinical trials in 2020 as both a monotherapy and in combination with anti-PD-L1 antibodies. This approach appears to be the most promising for all those patients who have developed an endogenous T cell response to the tumor. Also, it is widely accepted that, to obtain high and superior therapeutic responses, in addition to stem-like CD8⁺ T cell-mediated responses, innate NK-type effectors and M1-type macrophages are also necessary. Indeed, the combination treatment with the anti-PD-L1 and muPD1-IL2v supported the immune response by reprogramming tumor-associated macrophages and increasing the diversity of the immune repertoire [128]. In particular, single cell RNA sequence analyses of myeloid cells showed that, in tumor-bearing mice treated with the aforementioned combination therapy, there was a reduction in the expression of M2-like immunosuppressive markers such as Trem2, Mrc1, and CD163, alongside an upregulation of pro-inflammatory markers such as Irf1, Slamf8, IFNγ, as well as the presence of T cell chemokines like as CXCL9 and CXCL10 [128]. Furthermore, in the orthotopic glioma model with the GL261 cells, it has been reported that the combination treatment can increase stem-like PD-1⁺TCF-1⁺CD8⁺ T cells, as well as their progeny, PD-1⁺TCF-1^- and effector CD8⁺ T cells. In this model, CD8⁺ T cells isolated from mice treated with the combination were less suppressive than those isolated from mice treated with the either of the two drugs alone. Again, a shift from M2 to M1 macrophages was detected. These findings suggested that, in several different mouse models, the combination of ICI and IL-2 variants can contribute concur to an optimal anti-tumor immune response [128]. Besides the effects described, it is worth to note that the combination treatment can affect the phenotype of endothelial cells present within the tumor. Indeed, these endothelial cells can directly regulate the infiltration, proliferation, and cytotoxicity of CD8⁺ T cells in an antigen specific manner [131,132].

6. Role of Radiotherapy in the Triggering Anti-Tumor Therapeutic Responses in Combined Tumor-Targeted IL-2 Treatments

Recent research has shown a growing interest in investigating the synergistic effects of combining radiotherapy (RT) with tumor-targeted IL-2 treatments to eradicate tumors. Indeed, the relationship between RT and the modulation of the immune system response have revealed novel regulatory pathways [133,134]. These include the concept of radiation-induced tumor equilibrium (RITE), which serves as a starting point to discuss the mechanistic influence of immune-checkpoint therapies on the efficacy of RT [133,134].

Traditionally viewed as a local therapy, RT directly damages DNA in tumor cells [135]. RT usually induces the release of danger signals and consequently chemokines that recruit inflammatory cells into the TME, including antigen-presenting cells that activate cytotoxic T-cell function. However, RT also has the capability to attract immunosuppressive cells into the TME [136]. Consequently, it has been shown that RT exhibits both immunostimulatory and immunosuppressive properties, owing to the inherent sensitivity of immune cells to its effects [137,138].

Concerning immunostimulatory effects, localized RT can provoke systemic immune responses [139] by inducing the expression of tumor-associated antigens and generating new tumor antigens that can activate antitumor immune responses. This helps counter the tumor's suppression of antigen presentation. For example, the expression of MHC-I, a critical antigen recognized by CD8⁺ antigen specific T cells, is often decreased on tumor cells [140]. RT can effectively increase MHC-I expression, facilitate dendritic cell maturation and leukocyte infiltration of tumors [141], reduce the presence of Treg cells within tumors, expand T-cell populations, and enhance T cell migration. Moreover, RT has demonstrated the capability to convert non-immunogenic tumors into immunogenic ones, either partially or completely [142]. Previous studies using IL-2 immnocytokine, such as L19-IL-2, have shown that it can synergize with RT to achieve tumor rejection and immune-mediated protection effect in a colon carcinoma mouse model [143,144,145]. Additionally, RT can manage local tumor progression when combined with anti-PD1/PDL1 or anti-CTLA4 ICI, immunocytokines, dendritic cell vaccines, and Toll-like receptor antagonists. These therapeutic combinations improve the overall survival (OS) and elicits specific immune responses against cancer [146]. Some recent preclinical and clinical research has demonstrated that the combination of RT’s immunosuppressive and immunostimulatory properties can lead to abscopal effects [147] and potentially a "radio-memory" effect, wherein the synergistic action of combo treatments produces enhanced outcomes [148].

Investigating whether the "radio-memory" effect [149] extends beyond anti-PD1/PDL1 ICI to other forms of immune therapy, such as IL-2, is a critical step in understanding the broader implications of RT-induced immune responses in cancer treatment. For instance, in patients with malignant pleural effusion who had previously received RT for non-small-cell lung cancer within 18 months, intrapleural infusion of IL-2 or cisplatin demonstrated potential evidence of a radio-memory effect [149].

More recently, other studies have examined the critical role of IL-2 in synergistic immunotherapeutic strategies involving RT. In particular, a modified IL-2-based treatment has shown promising results when combined with RT to amplify immune responses. Gadwa and colleagues demonstrated that combining RT with PD1-IL2v, an IL-2 variant targeting intermediate-affinity IL-2 receptors (IL-2Rβγ), significantly enhanced immune activation; The lymphocytes involved are mainly CD8+ T cells and NK cells, while a concomitant reduction of the suppressive function of Treg cells was detected [150]. This combination showed effective tumor control and reduced metastasis, leveraging the immune system's response to control distant tumors via an "abscopal effect". In a parallel investigation, He et al. focused on an engineered IL-2 variant, RDB 1462, which binds selectively to IL-2Rβγ, promoting antitumor activity while minimizing the induction of Tregs [151]. Their findings revealed that combining high-dose RT with RDB 1462 significantly improved survival and suppressed primary and metastatic tumor growth in mouse models. Moreover, this combination therapy enhanced CD8⁺ memory T cells and NK cell activation, while diminishing Treg and MDSCs. The precise timing of IL-2 administration before RT was crucial in maximizing therapeutic efficacy and immune response [151]. These findings demonstrate that IL-2, particularly in its engineered forms, holds substantial promise when paired with RT to enhance anti-tumor immunity. By targeting specific IL-2 receptor subunits, these treatments can potentially overcome immune suppression within the TME and improve the therapeutic outcomes of RT and other immunotherapies. Further clinical investigations are warranted to optimize this approach for broader cancer treatment strategies (Figure 2).

7. Conclusions

The integration of IL-2 immunocytokines into cancer therapy marks a promising breakthrough, overcoming the challenges associated with traditional IL-2 treatment. While high-dose IL-2 has shown efficacy in specific cancers, the severe toxicities have limited a broader use. The targeted delivery of IL-2 through immunotoxin and immunocytokines, in combination with checkpoint inhibitors, chemotherapy, radiotherapy, and anti-angiogenic agents, has significantly enhanced anti-tumor responses while reducing toxicity. These strategies capitalize on the dual activation of innate and adaptive immune responses, effectively counteracting immune suppression and improving tumor clearance.

Innovations such as antibody fusion proteins, modified IL-2 variants, and bi-specific formats further optimize the targeting of the TME, improving therapeutic efficacy without inducing significant side effects. The use of PD-1-targeting IL-2 immunocytokines, in particular, has demonstrated the ability to rejuvenate exhausted T cells and enhance immune infiltration within tumors, offering substantial promise in both primary and metastatic cancer settings. Moreover, advances in cytokine engineering, including glyco-engineering and prodrug formulations, have enhanced the selectivity and cost-effectiveness of these therapies, ensuring their viability in diverse clinical contexts.

Looking ahead, continued research into IL-2 immunocytokines holds great potential for developing therapies that not only target primary tumors but also overcome metastasis and therapy resistance. As these treatments evolve, the ability to precisely modulate immune responses on demand could redefine cancer therapy, offering durable responses and improved survival outcomes for patients worldwide. Further exploration in clinical settings is crucial to unlock the full potential of IL-2 immunocytokines, ensuring their effective translation from preclinical models to routine clinical use.