Therapeutic application of IL-12 for cancer therapy

Introduction

Cancer accounts for 16.8% of deaths globally, making it the second leading cause of death, and a crucial public health issue worldwide in the 21st century (1,2). However, conventional therapies such as surgery, chemotherapy, molecularly targeted therapies, and radiation often yield inconsistent outcomes (3,4). Immunotherapy augments and reprograms the patient’s immune system to recognize and eliminate cancer cells and has emerged as a critical approach in the treatment of certain cancer types (5). However, several challenges in targeting solid tumours—including an immunosuppressive tumour microenvironment (TME), poor immune cell trafficking and tumour infiltration, and difficulties in tumour target selection—have limited success compared to haematological malignancies (6,7). Cytokine immunotherapy represents a promising strategy to convey pro-inflammatory cues to the tumour site, thereby remodelling the TME and converting an immunologically ‘cold’ tumour into a ‘hot’ one (7,8). This objective led to the investigation of interleukin-12 (IL-12), a potent pro-inflammatory cytokine with various anti-tumour functions. Kobayashi and colleagues first identified IL-12, which they designated as natural killer (NK) cell stimulatory factor (9). IL-12 is a p70 heterodimer consisting of the p35 (α-chain) and p40 (β-chain) subunits covalently linked by disulfide bonds (10). The IL-12 receptor (IL-12R) is also composed of two subunits, IL-12Rβ1 and IL-12Rβ2, and is expressed on both innate and adaptive immune cells, including dendritic cells (DCs), NK cells, activated T cells and B cells (11-13). Unlike receptors with intrinsic catalytic activity, the IL-12R depends on members of the Janus kinase (JAK) family to initiate intracellular signalling cascades (14). Upon binding to its receptor, IL-12 activates the downstream JAK/signal transducer and activator of transcription (STAT) pathway (15). The receptor-associated JAKs, Tyk2 and Jak2, which bind to the IL-12Rβ1 and IL-12Rβ2 subunits respectively, become activated through transphosphorylation and subsequently phosphorylate tyrosine residues on the intracellular domains of the IL-12R (16,17). These phosphotyrosine sites serve as docking platforms for STAT transcription factors, which are recruited via their SH2 domains (18). Once engaged, STATs are tyrosine-phosphorylated by JAKs, leading to their dimerization, nuclear translocation, and subsequent regulation of gene expression through interaction with specific DNA sequences (19). While IL-12 has been shown to activate STAT1, STAT3, STAT4, and STAT5, STAT4 is the principal transcription factor that mediates downstream signalling (20). Notably, STAT4 drives transcription of the gene encoding interferon-gamma (IFN-γ) (20), a critical cytokine that acts on IL-12-producing antigen-presenting cells (APCs) to further enhance IL-12 secretion (21), thereby establishing a positive feedback loop (22).

IL-12 acts as a bridge between innate and adaptive immunity. APCs, such as DCs and macrophages, are the primary producers of IL-12 in response to stimuli such as microbial infection (23,24). Secreted IL-12 exerts its anti-tumour effects by inducing the activation, proliferation and cytolytic activity of T cells and NK cells (25,26). Furthermore, IL-12 can drive the differentiation of naïve CD4⁺ T cells into T helper type 1 (Th1) cells, thereby initiating a pro-inflammatory immune response (27,28). Given these findings, IL-12 has been investigated as an experimental cancer immunotherapy. In 1993, the intraperitoneal administration of recombinant IL-12 was first evaluated in several murine tumour models and demonstrated a robust anti-tumour effect (29). However, while potent efficacy was also seen in haematological malignancies (30), early human clinical trials showed that systemic administration of IL-12 resulted in severe toxicity (10).

In the first phase I human study in 1997, the 500 ng/kg intravenous dose level was determined to be the maximum tolerated dose (MTD) (31). However, in a subsequent phase II trial, 12 of 17 patients who received the MTD experienced severe adverse events such as leukopenia (65%), hyperbilirubinemia (47%), and elevated aspartate aminotransferase levels (47%), and two patients died (32). The significant difference in toxicity between the two studies was attributed to a change in the IL-12 injection schedule; specifically, that a single test dose of IL-12 was administered 14 days prior to the repeated dosing in the phase I study, but not in the phase II study (10,32). In conjunction with the limited clinical responses observed in other phase II trials, interest in IL-12-based cancer immunotherapy waned (33,34). Yet, these early lessons laid the conceptual framework for the design of tumour-targeted and controllable IL-12-based therapeutics that have been developed more recently (20,35). Over the last decade, the swift emergence of other immunotherapies that primarily rely on the activation and efficacy of T cells and NK cells, including immune checkpoint inhibitors (ICIs) and adoptive cell therapy (ACT) (5), has transformed the landscape of cancer treatment. Together with modern advances in molecular engineering technologies, enthusiasm for IL-12-based immunotherapy has been rekindled (7).

This review examines the immunomodulatory role of IL-12 in enhancing anti-tumour immunity and assesses its therapeutic potential in cancer, both as a single agent and combination therapy. Although IL-12 has shown promise in both haematological and solid malignancies (30,36,37), the majority of translational advances and delivery innovations have involved solid tumours, which are the primary focus of this review.

Anti-tumour activities of IL-12

IL-12 is a pleiotropic cytokine that plays a vital role in initiating and coordinating anti-tumour immune responses (10). Several mechanisms underlie its anti-tumour activity (Figure 1), the first being the promotion of activation, proliferation and cytotoxic activity of NK cells and CD8⁺ cytotoxic T lymphocytes (CTLs) (26,38). These effector cells play a crucial role in eliminating malignant cells via granule-mediated cytotoxicity, which depends on the release of the pore-forming protein perforin and the serine protease granzyme B from cytoplasmic lytic granules (39-44). In addition to the well-known roles of CTLs and NK cells, anti-tumour activity of NK T (NKT) cells is also enhanced by IL-12 (45). The potent anti-tumour effects induced by IL-12 are chiefly mediated by the effector cytokine IFN-γ, which is produced by NK cells, T cells, and NKT cells upon stimulation with IL-12 (46). IFN-γ can also trigger APCs to enhance IL-12 production, resulting in a positive feedback loop (47).

Figure 1 Anti-tumour mechanisms induced by IL-12. Created with BioRender.com. IFNγ, interferon gamma; IL-12, interleukin-12; MDSC, myeloid-derived suppressor cell; MHC, major histocompatibility complex; NK, natural killer; TCR, T-cell receptor; Th, T helper cell.

IFN-γ is a multifunctional cytokine that engages in various anti-tumour functions via its receptor (IFNGR) (48), thereby orchestrating both innate and adaptive immune responses (49). It stimulates APCs such as DCs, leading to their maturation and upregulation of major histocompatibility complex (MHC) class I and II complex expression levels, thereby facilitating the presentation of tumour antigen to T cells (49-51). It is also a vital effector cytokine of the Th1 response. When secreted by IL-12-polarized CD4⁺ Th1 cells, IFN-γ establishes a self-amplifying circuit to promote its own secretion, inhibit differentiation into Th2 and Th17 phenotypes (52), while stabilising the proinflammatory Th1 cell phenotype (53). Moreover, IFN-γ boosts the cytotoxicity of killer lymphocytes, including NK cells and cytotoxic CD8⁺ T cells, further facilitating tumour control (54,55).

IFN-γ also contributes to anti-tumour efficacy through its actions on non-immune cells. Thus, IFN-γ has been reported to activate JAK-STAT1-caspase signalling in non-small cell lung cancer (NSCLC) cells, leading to their apoptosis (56). In addition, IFN-γ can compromise tumour growth by inhibiting angiogenesis, a process essential for the progression of solid tumours (57). In addition to induction of IFN-γ secretion, IL-12 stimulates T cells and NK cells to produce several other proinflammatory cytokines, including TNF-α, granulocyte-macrophage colony-stimulating factor (GM-CSF), and IL-2 (58).

Myeloid immune cells within the TME, such as myeloid-derived suppressor cells (MDSCs) and tumour-associated macrophages (TAMs), actively contribute to tumour progression and metastasis by sustaining an immunosuppressive milieu (59). IL-12 drives the polarisation of TAMs from a tumour-supportive M2 phenotype—marked by the production of monocyte chemotactic protein-1, IL-10, and transforming growth factor-beta (TGF-β)—to a pro-inflammatory and anti-tumour M1 phenotype that secretes immune-activating cytokines such as IL-1, IL-15, and TNF-α (60). IL-12 also modulates the phenotype and function of MDSCs by reprogramming their immunosuppressive activity. Upon exposure to IL-12, MDSCs upregulate the expression of MHC class II and CD86 (61), which respectively enhance antigen presentation and T cell co-stimulation. Additionally, IL-12-treated MDSCs exhibit reduced production of nitric oxide, a key immunosuppressive mediator, thereby diminishing their ability to suppress T-cell activity (62,63).

Lastly, a critical anti-tumour mechanism mediated by IL-12 is epitope or antigen spreading. This refers to an immune response that initially targets a specific tumour antigen but subsequently broadens to encompass additional antigens (64). When IL-12 is delivered into the TME, it enhances the cytotoxic activity of antigen-specific T cells, leading to tumour cell lysis (65). This process releases non-targeted tumour antigens, which are subsequently captured by IL-12-activated APCs and presented to prime naïve endogenous CD4⁺ and CD8⁺ T cells. By this means, newly activated T cells recognize secondary antigens, expand and infiltrate the tumour lesion (66).

Strategies for safer IL-12 delivery in cancer therapy

Systemic administration of IL-12 triggers rapid elevations of pro-inflammatory cytokines, including IFN-γ, TNF-α, GM-CSF, and IL-6, a phenomenon referred to as cytokine release syndrome or “cytokine storm” (10,67,68). This systemic cytokine surge drives widespread inflammation that leads to organ injury, such as hepatotoxicity manifested by elevated serum transaminases (46). It is often accompanied by leukopenia and thrombocytopenia resulting from consequent immune suppression, together contributing to multi-organ failure and constituting a potentially lethal process in humans (69).

The severe systemic toxicities observed in early human clinical studies fundamentally influenced the later design of IL-12-based therapeutics. Patients enrolled in clinical trials receiving systemic IL-12 experienced adverse events, including abnormal laboratory parameters (e.g., elevated IFN-γ levels, leukopenia, and increased transaminases) and clinical symptoms (e.g., fatigue, dyspnoea, and stomatitis) (31,32). These findings revealed that uncontrolled systemic IL-12 dissemination represented a fundamental barrier to its therapeutic translation and emphasised the need to spatially and temporally confine cytokine activity (70). Accordingly, to effectively harness IL-12’s robust biological activities, subsequent design strategies shifted focus toward tumour-targeted delivery to confine IL-12 biological activity within the TME (20,71). Two main strategies have been employed, entailing the use of fusion proteins called immunocytokines and engineered immune cells.

Immunocytokines

Immunocytokines are fusion molecules in which a tumour-targeting antibody or derived fragment is joined to a cytokine, such as IL-12 (72). By leveraging antibodies that recognise antigens overexpressed or uniquely present within tumour lesions, immunocytokines can localise more precisely to the TME than unmodified cytokines (73).

Among IL-12-based immunocytokines, NHS-IL12 is the most comprehensively investigated candidate. It has progressed significantly in clinical development, with several trials evaluating its therapeutic potential across various malignancies (71). NHS-IL12 is composed of a full-length human IgG1 monoclonal antibody (NHS76) genetically fused to two human IL-12 heterodimers, which are attached to the C-terminal end of each heavy chain of the antibody (Figure 2) (74).

Figure 2 A schematic of NHS-IL12 interacting with exposed nucleic acids at the necrotic tumour site. Created with BioRender.com. IL-12, interleukin-12.

Solid tumours often contain necrotic regions due to insufficient vascularisation, hypoxia and limited nutrient supply. NHS76 binds to exposed nucleic acid/histone epitopes within these areas, thereby serving as an effective vehicle for directing IL-12 to the tumour site (75,76). Evaluating the in vivo activity of IL-12-based immunocytokines has been challenging because human IL-12 lacks biological activity in mice. To enable the assessment of anti-tumour efficacy, a murine analogue of NHS-IL12 was developed by fusing the human NHS76 antibody with two murine IL-12 heterodimers (77). In preclinical murine tumour models, the NHS76-murine IL-12 fusion protein (NHS-muIL12) exhibited greater anti-tumour activity compared to recombinant IL-12. It effectively localized to subcutaneous tumours in vivo and significantly outperformed an equivalent concentration of recombinant murine IL-12 in suppressing tumour growth (76). Fallon and colleagues found that NHS-muIL12 induced dose-dependent increases in the expression of MHC-I complex on DCs, raised serum IFN-γ levels and enhanced both NK cell and CD8⁺ T cell proliferation. Using immune cell subset–depleting antibodies, they showed that the anti-tumour efficacy of NHS-muIL12 relied primarily on CD8⁺ T cells and, to a lesser extent, on NK cells (76,78).

Collectively, these early findings laid the foundation for advancing NHS-IL12 into clinical development. In the first-in-human phase I trial of NHS-IL12, NHS-IL12 was well tolerated at 16.8 mg/kg MTD. Pharmacodynamic data from all subjects (single and multiple doses) showed a temporal increase in IFN-γ, followed by a subsequent increase in IL-10 (79). In most patients receiving multiple doses, serum IL-12 levels rose consistently; however, the subsequent increases in IFN-γ and IL-10 were attenuated after the second dose. A similar pattern was observed for IFN-γ-inducible chemokines, including IFN inducible protein 10 and CXC chemokine ligand 10 (79). These findings suggest that later doses of NHS-IL12 exhibited improved tolerability associated with reduced systemic toxicity compared to the initial dose. This effect might be attributable to a cytokine “priming” phenomenon previously seen in earlier studies with recombinant IL-12 monotherapy, and may offer a strategy to dampen down toxicity in prospective treatment regimens (31,32,80). Moreover, NHS-IL12 treatment enhanced T-cell receptor (TCR) diversity and increased tumour-infiltrating lymphocyte (TIL) density in subjects with high IFN-γ responses, indicating enhanced T-cell recruitment into the TME (79,80). As a result, NHS-IL12 helped to convert non-immunogenic (“cold”) tumours into immunologically responsive ones, thereby broadening the range of cancers that could benefit from immune checkpoint blockade. Accordingly, NHS-IL12 has been further investigated in combination with checkpoint inhibitors although results have been disappointing. In the first-in-human phase Ib trial, M9241 (NHS-IL12), in combination with the anti-programmed cell death ligand 1 (PD-L1) checkpoint inhibitor avelumab, was administered subcutaneously to patients with advanced bladder tumours. However, among the 36 participants, only two subjects achieved a complete response (81). In the planned two-stage dose-expansion phase of the study conducted in patients with advanced urothelial carcinoma (UC), the combination of NHS-IL12 and avelumab was well-tolerated but no objective responses were recorded in stage one (16 subjects). Consequently, the study did not proceed to stage 2, as it did not meet the predefined standard of at least three confirmed objective responses (81). The failure to demonstrate a synergistic benefit from combining NHS-IL12 with anti-PD-L1 was unexpectedly disappointing and did not align with previous reports of avelumab monotherapy, which showed an objective response rate of 16.5% in advanced UC (82). It was speculated that this may have resulted from an IL-12-induced negative feedback loop, whereby IFN-γ upregulated PD-L1 both within the TME and systemically-potentially serving as a sink for avelumab (83). Furthermore, the increased expression of checkpoint receptors, such as LAG-3, suggested that IFN-γ may have initiated other immunosuppressive pathways (81). In this case, the dosing schedule of avelumab may have been insufficient to overcome elevated PD-L1 levels and unable to counteract alternative suppressive mechanisms beyond the PD-L1 axis (81). This highlights a key limitation of IL-12-based therapies, namely that IL-12 can intensify immune exhaustion through sustained IFN-γ production (7).

Beyond targeting necrotic tumour regions, immunocytokines have also been designed to engage various additional tumour-specific features. One example is the transmembrane glycoprotein epithelial cell adhesion molecule (EpCAM), a widely expressed tumour-associated antigen (TAA) found in many epithelial-derived cancers (84). HuKS-IL-12 is an immunocytokine that links IL-12 to the Fc domain of a humanized antibody directed against EpCAM. In a mouse prostate cancer xenograft model, HuKS-IL-12 demonstrated strong efficacy by significantly reducing established lung metastases in SCID mice that had been transplanted with activated human T cells and NK cells (85). Another TAA, ganglioside GD2, is a glycolipid mainly expressed in neuroblastoma tumours and sarcomas, and has been targeted using an immunocytokine named Hu14.18-IL-12 (85,86). A major challenge in targeting TAAs arises from intra- and inter-tumoural heterogeneity resulting from mutations and epigenetic instability. Moreover, since only a subset of patients may express the target of interest, this may further limit the therapeutic application to a specific patient group (73,87).

A novel immunocytokine has been engineered by fusing the A3 collagen-binding domain (CBD) of the glycoprotein von Willebrand factor (VWF) to each subunit of IL-12 (88). Ishihara and colleagues reported that following intravenous administration, the CBD of VWF accumulated in solid tumours due to the leaky tumour-associated vasculature, which exposes collagen locally. This is amplified by the fact that collagen is more abundant in the tumour stroma than in healthy tissues (89). In mouse models of melanoma and breast cancer, the systemic administration of CBD-IL-12 fusion demonstrated greater tumour localisation and antitumour activity than unmodified IL-12. Although the breast cancer model was poorly infiltrated by CD8⁺ T cells (89), treatment with CBD-IL-12 enhanced this, effectively transforming an immunologically ‘cold’ tumour into a ‘hot’ phenotype. Moreover, the fusion of CBD with IL-12 helped to reduce systemic toxicity. Studies using IFN-γ receptor-deficient mice demonstrated that the majority of IL-12-associated adverse effects are mediated by IFN-γ. CBD-IL-12 appreciably reduced circulating levels of IFN-γ over multiple dosages in comparison with IL-12 (88).

Although the immunocytokine approach can increase the specificity of tumour delivery while maintaining reduced toxicity, it is unlikely to eliminate safety concerns entirely. As noted earlier, these agents may still interact with circulating immune cells and tissues, triggering downstream signalling while moving from the injection site to the TME (81).

ACT

T lymphocytes perform immune surveillance by recognising antigens presented on MHC molecules. In each case, antigenic specificity is conferred by the clonotypic TCR expressed on the cell surface (90,91). Nonetheless, transformed tumour cells can acquire mechanisms to evade detection by T cells, such as reducing the expression or processing of tumour antigens, while solid tumours frequently develop an immunosuppressive microenvironment that hinders T cell activity and impairs their cytotoxic function (92). ACT encompasses the use of ex vivo expanded TIL cells, TCR-engineered T cell therapy and chimeric antigen receptor (CAR)-engineered T cell therapy, all of which have demonstrated promising benefits in diverse types of cancer (93).

A CAR is a genetically encoded synthetic receptor which couples the MHC-independent binding of a cell surface target to the delivery of a tailored T cell activating signal (94-96). The most remarkable outcomes of CAR T-cell immunotherapy have been achieved using CD19⁺ and B cell maturation antigen targeted CAR T-cell therapies in patients with B-cell malignancies and myeloma, respectively. Currently, there are seven Food and Drug Administration (FDA)-approved CAR T-cell therapies for blood cancers (97,98). However, the same clinical success has not been seen with solid tumours owing to the lack of tumour-selective and uniformly expressed antigens, difficulty in accessing and infiltrating solid tumour deposits and the immunosuppressive TME, which promotes T-cell exhaustion. To address this, engineered T cells have been modified to co-express immunomodulatory cytokines such as IL-12. By this means, the intention is to favourably reshape the TME, making it more conducive to T-cell activity and enhancing the recruitment and function of ACT cells (94).

IL-12 has been incorporated into ACT products using a range of strategies. Illustrating this, TCR-engineered T cells were co-engineered to secrete a single-chain IL-12 cytokine fusion. In mice bearing B16 melanoma, administration of IL-12 armoured TCR-engineered T cells effectively eradicated large, established tumours following lymphodepletion. The engineered cells secreted IL-12 directly into the tumour site at therapeutic levels, augmenting their activity and promoting greater tumour infiltration of both the transferred cells and endogenous NK and CD8⁺ T cells when compared to non-transduced T cells (99). However, IL-12-induced toxicities, including weight loss and reduced survival, were observed in mice injected with more than 500,000 cells (99). This finding may be explicitly attributed to the elevated systemic level of IL-12 generated through its constitutive production by the engineered T cells (99). Accordingly, to mitigate the toxic effects resulting from sustained IL-12 expression, researchers constructed a nuclear factor of activated T cells (NFAT) promoter-inducible IL-12 cassette and introduced it into TCR-engineered and CAR T cells (Figure 3) (100,101). The goal of this approach was to ensure that biologically active IL-12 was released exclusively when the TCR or CAR engaged with the cognate antigen, selectively triggering T-cell activation within the TME (100). NFAT is a vital transcription factor that is induced by T-cell activation through the Ca²⁺-calcineurin signalling pathway (102,103). When mice with B16 melanoma were treated with NFAT-IL-12 armoured T cells that co-expressed a TCR targeted against gp100 or a CAR directed against vascular endothelial growth factor receptor 2, T cells infiltrated tumours more efficiently, achieved prolonged persistence, and elicited sustained tumour regression without notable toxicity (100,101). Therefore, the toxicities caused by constitutive IL-12 expression could be mitigated while preserving efficacy.

Figure 3 A schematic of Ca⁺-calcineurin signalling-mediated NFAT-inducible IL-12 secretion in T cells upon CAR or TCR engagement. Created with BioRender.com. CAR, chimeric antigen receptor; IL-12, interleukin-12; MHC, major histocompatibility complex; NFAT, nuclear factor of activated T cells; TCR, T-cell receptor.

Zhang and colleagues conducted a first-in-human phase I clinical trial to engineer autologous TIL cells with NFAT-inducible IL-12 to treat patients with metastatic melanoma. Treatment with this modified TIL approach achieved tumour reduction while requiring dramatically fewer cells—50 to 100 times less—than required with conventional TIL therapies. Notably, objective responses were observed in 63% of patients (10 out of 16) who received doses of between 0.3×10⁹ and 3×10⁹ cells (104). Nonetheless, patients who received cell doses sufficient to induce tumour reduction experienced substantial toxicity, including liver dysfunction and fever due to elevated serum concentrations of IL-12 and IFN-γ. This was attributed to leakiness of the inducible system. Thus, to retain the advantages of localized expression while limiting systemic activity, it is important to minimize circulating IL-12 levels (104). This principle guided the development of membrane-anchored IL-12 (maIL-12) (Figure 4), where IL-12 is tethered to the T-cell surface through fusion with a suitable transmembrane domain, such as derived from EGFR, CD28, or B7.1 (70,105,106).

Figure 4 CAR T-cell expressing a membrane-anchored IL-12. Created with BioRender.com. CAR, chimeric antigen receptor; IL-12, interleukin-12.

Membrane-anchored IL-12 was co-expressed in CAR-T cells targeting the TAA TAG72 (TAG72-CAR/maIL-12). It was shown that maIL-12 acted both on host T cells (in cis) and on neighbouring T cells (in trans). The authors developed an ovarian cancer (OV90) dual-tumour xenograft model featuring both loco-regional disease [e.g., intraperitoneal (i.p.) metastases] and systemic disease, modelled by the introduction of subcutaneous (s.c.) tumours in the mice. They observed that i.p. delivery of maIL-12-engineered CAR T cells led to superior tumour reduction at both local and distant sites compared to treatment with TAG72 CAR T cells alone. Next, an immunocompetent mouse model using murine ID8 ovarian tumour cells expressing TAG72 was established to test the safety of this maIL-12 engineered CAR T-cell approach. Co-administration of TAG72-CAR T cells and soluble IL-12 resulted in significant weight loss, splenomegaly, and liver abnormalities. In contrast, treatment with TAG72-CAR/maIL-12 T cells did not elicit detectable systemic adverse effects. Importantly, soluble IL-12 injections resulted in increases in systemic IFN-γ levels in the serum of TAG72-CAR T-cell-treated mice, which were undetectable in TAG72-CAR/maIL-12 treated mice. This approach also favourably modulated the immunosuppressive TME, as evidenced by enhanced IFN-γ production, T-cell infiltration, prolonged persistence of CD8⁺ T cells, and upregulated MHC-II expression on DCs, indicating a shift toward a mature APC phenotype (106).

In a study using an osteosarcoma patient-derived xenograft (PDX) tumour model, a tumour-targeted and membrane-anchored variant of IL-12 (ttmaIL-12) was co-expressed in B7H3-targeted CAR-T cells. Tumour targeting was achieved by addition to the p40 subunit of a short peptide (VNTANST), which binds vimentin expressed on the surface of numerous solid tumours. Membrane anchoring was maintained by linking the IL-12 p35 subunit to a transmembrane domain (107). The ttmaIL-12 B7H3-targeted CAR-T cells were robustly guided to tumour sites while limiting systemic cytokine dissemination. As a result, these cells achieved not only enhanced suppression of tumour growth but also produced a large amount of IFN-γ when present within the TME. IL-12-mediated toxicity was circumvented using the ttmaIL-12 technology via two mechanisms. First, accumulation of these cells outside of the tumour was minimised, in contrast to T cells that expressed soluble IL-12, maIL-12-T or no cytokine. Second, systemic cytokine exposure was also reduced.

In the same study, OVA-specific OT-1 TCR T cells armoured with ttmaIL-12 eradicated OVA-negative B16F10 melanoma tumours, whereas rapid tumour progression was observed in mice receiving OT-1 T cells or OT-1 T cells constitutively secreting IL-12. Tumour rejection was mediated by epitope spreading (105). Similarly, Kueberuwa and colleagues reported that murine IL-12-secreting anti-mouse CD19 CAR T cells could eliminate established A20 B cell lymphoma through a synergistic mechanism involving direct lysis of CD19⁺ tumour cells and activation of endogenous immune cells to elicit an anti-tumour response against antigens beyond CD19. Epitope spreading could play a crucial role in limiting tumour immune escape resulting from downregulation or loss of the cognate antigen required for the recognition of antigen-specific T cells (108).

Zhang et al. designed a therapeutic platform combining maIL-12 with an NFAT-responsive promoter, whereby TCR activation in T cells triggered maIL-12 expression. This strategy enabled tumour-targeted delivery of IL-12 by T cells while reducing systemic toxicity. The combination system was dubbed inducible anchored IL-12 (iaIL-12). In the B16 OVA tumour model, OT-1 TCR T cells transduced with iaIL-12 achieved superior efficacy compared with OT-I TCR T cells expressing NFAT-inducible green fluorescent protein (GFP). Mice treated with iaIL-12 showed neither weight loss nor reduced survival, in contrast to those receiving NFAT-inducible secreted IL-12 (isIL-12). Unlike isIL-12, treatment with iaIL-12 did not increase serum levels of IL-12, IFN-γ, IL-10, or TNF-α (109). To test iaIL-12 in human T cells, it was co-expressed with a TCR targeting human papillomavirus-16 viral oncoprotein E7. When compared with isIL-12 and constitutively anchored IL-12 (caIL-12), activated T cells engineered with iaIL-12 released markedly lower amounts of IL-12 into the culture supernatant, indicating that the molecule underwent minimal shedding and secretion. When tested in vivo in a cervical cancer xenograft model, E7-TCR T cells engineered with iaIL-12 achieved more pronounced tumour reduction compared with E7 TCR-T cells engineered to co-express inducible GFP (110). By contrast, isIL-12 but not iaIL-12 armoured cells induced markedly elevated serum IL-12, as a result of which mice either succumbed or required euthanasia beyond day 15 due to toxicity. In both murine tumour and human xenograft tumour models, iaIL-12 T cells displayed minimal systemic distribution and greater antitumour effect compared to isIL-12 and inducible GFP, respectively. One limitation of this combination system is the lack of precise regulation over iaIL-12 gene expression, with some basal NFAT-dependent expression occurring even in the absence of stimulation (109). Employing endogenous tumour-localized promoters to trigger transgene expression may offer superior regulatory precision than a synthetic promoter (111).

Recently, Chen and colleagues developed a novel approach to deliver IL-12 directly to tumours using CAR T cells. They screened for endogenous genes that were markedly upregulated in tumour-infiltrating CAR T cells compared to spleen, identifying NR4A2 as a top candidate (111). In mice bearing E0771-hHer2 tumours, GFP expression in murine anti-Her2 CAR T cells was regulated by either an NFAT-responsive promoter or via CRISPR knock-in to the endogenous NR4A2 promoter. CAR T cells engineered with the NFAT-inducible GFP showed substantial off-tumour GFP expression. In contrast, NR4A2-driven GFP expression remained tightly tumour restricted, with no more than 2% GFP⁺ cells observed elsewhere. IL-12 was then expressed using a similar approach by murine CAR T cells, linking its transcription to the endogenous NR4A2 promoter (NR4A2/IL-12) (Figure 5). Anti-hHer2 CAR T cells engineered with mock edited (unmodified NR4A2), NR4A2 knockout or IL-12 knock-in (NR4A2/IL-12) were infused into E0771-hHer2 breast carcinoma or MC38-hHer2 colon adenocarcinoma tumour-bearing mice. NR4A2/IL-12 murine CAR T cells exhibited a significantly improved anti-tumour response without toxicity. Conversely, E0771-hHer2 tumour-bearing mice treated with NFAT-inducible IL-12-expressing murine CAR T cells showed pronounced toxicity, manifested by swift loss of body weight, hunched posture, and reduced activity. A similar enhanced therapeutic response and safety profile was observed in a human xenograft OVCAR-3 ovarian cancer model, in which mice were treated with NR4A2/IL-12 human anti-LeY CAR T cells.

Figure 5 Schematic of a CAR T-cell engineered with the NR4A2-IL-12 system. IL-12 transgene expression occurs at the tumour sites where the endogenous gene is transcriptionally active. Created with BioRender.com. CAR, chimeric antigen receptor; IL-12, interleukin-12.

As noted above, previous research has highlighted the critical role of epitope spreading in facilitating a durable therapeutic effect following ACT (112). A marked increase in endogenous CD8⁺ T-cell infiltration was demonstrated in MC38-hHer2 tumours following treatment with murine anti-hHer2 CAR T cells engineered with the NR4A2/IL-12 system described above. Using tumour antigen-specific tetramers, it was shown that splenocytes from mice infused with NR4A2/IL-12 CAR T cells contained elevated numbers of tumour specific CD8⁺ T cells, when compared to mock and NR4A2 knockout controls. Additionally, mice bearing MC38-hHer2 tumours that were cured by NR4A2/IL-12 anti-hHer2 CAR T cells displayed a significant anti-tumour response upon rechallenge with hHer2-negative MC38 tumours, compared to untreated mice. This is consistent with the occurrence of epitope spreading to antigens beyond hHer2. It was also shown that the CRISPR knock-in strategy is applicable to patient-derived CAR T cells, indicating its promising clinical potential for tumour-restricted delivery of IL-12 (111).

Although maIL-12 expressed by CAR T cells can enhance therapeutic efficacy via epitope spreading while limiting systemic toxicities, its restricted cytokine release may result in more limited TME remodelling compared to soluble IL-12 (61,113). To address this, Murad et al. developed a CAR T-cell-engineered strategy to secrete a bifunctional fusion protein in which an anti-PD-L1 checkpoint inhibitor was fused to single-chain IL-12 (αPDL1-IL-12) (Figure 6) (114). The underlying rationale was to boost IFN-γ production, which in turn upregulated immune checkpoint ligands, such as PD-L1, within the TME. This allowed the anti-PD-L1 IL-12 fusion to be sequestered at the tumour site, boosting activity in a localised manner (115).

Figure 6 CAR T-cell engineered to produce a bifunctional fusion protein composed of αPDL1 checkpoint inhibitor and IL-12. Created with BioRender.com. CAR, chimeric antigen receptor; IL-12, interleukin-12.

To test the function and safety of this system, mice were injected with PTEN-Kras human prostate stem cell antigen (PSCA)-expressing mouse prostate tumour cells. Intravenous injection of PSCA-targeted CAR/αPDL1-IL-12 achieved the most potent therapeutic effect, resulting in complete tumour eradication in all mice. This significantly outperformed treatment using PSCA-CAR T cells alone, PSCA-CAR engineered with αPDL1 (CAR/αPDL1), or αPDL1^mut-IL-12 (CAR/αPDL1^mut-IL-12) in which αPDL1^mut could no longer bind to PD-L1. Notably, mice that received PSCA-CAR/αPDL1-IL-12 therapy maintained stable body weight, whereas those treated with either PSCA-CAR/αPDL1^mut-IL-12 or PSCA-CAR plus soluble IL-12 experienced marked weight loss, reflecting systemic toxicity (114). Moreover, PSCA-CAR/αPDL1^mut-IL-12 T cells treated mice demonstrated markedly elevated systemic IFN-γ levels compared to those treated with PDL1-IL-12-secreting counterparts. This indicates that PD-L1 engagement within the TME had constrained the systemic dissemination of IL-12 (114).

To extend these findings to a second model, mice were engrafted with ID8 murine ovarian cancer cells engineered to express the TAA TAG72. Treatment with TAG72-CAR T cells engineered to secrete either αPDL1^mut-IL-12 or αPDL1-IL-12 fusion proteins exhibited elevated infiltration of CD3⁺, CD4⁺, and CD8⁺ T cells compared to those treated with TAG72-CAR T cells alone. Importantly, tumours from both the αPDL1^mut-IL-12 and αPDL1-IL-12 groups showed substantially raised PD-L1 expression, potentially driven by enhanced local IFN-γ signalling. Furthermore, treatment with TAG72-CAR/αPDL1-IL-12 T cells led to a pronounced upregulation of proteins associated with lymphoid lineage cells (CD4, CD8), immune checkpoint molecules [programmed cell death 1 (PD-1), LAG3], and T cell activation (CD28, perforin) within the tumour. In contrast, these findings were substantially less pronounced in mice treated with conventional CAR T cells alone, in addition to those treated with CAR/αPDL1^mut-IL-12 or CAR/αPDL1 T cells. In addition, treatment with TAG72-CAR/αPDL1-IL-12 resulted in a significant decrease in CD14⁺ myeloid cells and expression of the suppressive CD163 M2 macrophage marker, but promoted an increase in CD11c⁺ DCs and MHC-II expression within the tumour compared to all other treatment groups (114,116). Using bifunctional PDL1-IL-12 fusion proteins as immunoregulatory agents represents a strategy applicable to a broad spectrum of solid tumours.

In 2024, Feng’s group also applied a strategy combining immunocytokines with CAR T-cell therapy. They engineered mesothelin (MSLN)-targeted CAR-T cells capable of secreting an immunocytokine (IL-12R54), consisting of single-chain IL-12 fused to an R54-derived scFv which binds a distinct MSLN epitope from that targeted by the CAR (117). In a KLM-1 pancreatic cancer xenograft model, MSLN-CAR-T cells engineered to secrete IL-12R54 (CAR-IL12R54) demonstrated markedly enhanced efficacy, extended survival, and elicited no detectable organ toxicity compared with CAR-T alone and CAR T cells that secreted unmodified IL-12. Furthermore, combining CAR-IL12R54 T cells with an anti-PD-1 antibody produced a synergistic anti-tumour effect accompanied by enhanced survival. Body weight changes did not differ significantly between mice receiving combination therapy and those treated with CAR-IL12R54 T-cell monotherapy. CXCL16 was identified as the predominant chemokine ligand produced by KLM-1 pancreatic cancer cells and was shown to enhance tumour cell proliferation, migration, and invasion. In vitro assays demonstrated that IL-12 promoted T-cell migration toward CXCL16-expressing tumour cells via NF-kB-mediated upregulation of CXCR6, the cognate chemokine receptor for CXCL16. As a result, T-cell infiltration and anti-tumour efficacy were both enhanced. A limitation of the study was the fact that safety could not be adequately addressed due to species-dependent differences in the biological activity of human IL-12 in immunodeficient mice (117).

Beyond the CXCL16-CXCR6 axis, additional chemokine-receptor pairs may also contribute to enhancing T cell infiltration into solid tumours. Zhang’s group demonstrated that the CCL4/CCL5-CCR5 axis is closely linked to T cell infiltration in multiple solid tumour types (118). Both CCL4 and CCL5 interact with CCR5, and tumours with elevated expression of CCL4 or CCL5 showed enhanced recruitment of CD8⁺ T cells, DC cells and NK cells. In contrast, tumours with lower chemokine expression had weaker immune cell infiltration (119). To improve the migratory capacity of CAR T cells, a MSLN-specific CAR was engineered to co-express CCR5 and IL-12 (CARTmeso-5-12). In a murine model, mice were subcutaneously implanted with human KYSE-510-CCL5 oesophageal cancer cells, which overexpress CCL5, along with M2 macrophages. Treatment with CARTmeso-5-12 cells led to a significant enhancement in T cell infiltration and a substantial reduction in the proportion of M2 macrophages compared to CD19-CAR T cells (control) and MSLN-targeted CAR T cells alone. CARTmeso-CCR5-IL-12 cells improved tumour infiltration through CCR5 overexpression, while IL-12 secretion reprogrammed TAMs, suppressing the M2 phenotype and promoting M1 polarisation. This dual action helped reverse the immunosuppressive TME, boosted CAR T-cell proliferation, function and infiltration, and collectively led to more effective tumour clearance (118). Once again, the safety of this approach would require evaluation in immune competent single species model systems.

Other IL-12 delivery methods

Besides immunocytokines and ACTs, alternative systems have been used to selectively deliver IL-12 as a therapeutic agent for cancer. Local injection of IL-12 often disseminates quickly into the circulation and causes systemic toxicity. Hence, biodegradable and biocompatible polymeric microspheres encapsulating IL-12 that are injected directly into the tumour site for sustained release have been investigated recently (120). Artificial polymers such as polylactic acid (PLA) are now being repurposed to encapsulate and deliver cytokines like IL-12 to tumours (120-123). For example, in BALB/c mice bearing established Line-1 lung alveolar carcinomas, intratumoural administration of IL-12-loaded PLA microspheres led to complete tumour regression in 53% of cases (8 in 15). Conversely, intratumoural delivery of free IL-12, at a dose equivalent to that administered via microspheres, achieved complete tumour regression in only 20% of mice (1 in 5) (122). However, cytokine-loaded polymeric microspheres have not been evaluated in clinical studies as yet. Although sustained intratumoural cytokine release is intended to localise IL-12 to the TME, a portion of the administered cytokine is still likely to disseminate into the bloodstream.

Comparative evaluation of IL-12 delivery strategies

Overcoming the toxicity associated with systemic IL-12 administration has led to the development of diverse delivery strategies aimed at restricting IL-12 activity to the TME. These approaches differ in their respective strengths and limitations, including the two principal categories discussed in this review, namely immunocytokines and ACT (7,10).

Immunocytokine platforms such as NHS-IL12 represent a promising approach for tumour-targeted IL-12 delivery and currently dominate the clinical landscape, reflecting greater translational maturity than ACT (35,70,71,124). However, they still permit measurable systemic exposure en route to the tumour site (125). In contrast, ACT-based IL-12 approaches offer greater spatial control, potentially reducing systemic toxicity through antigen-dependent IL-12 expression (e.g., NFAT-inducible systems) and a range of sophisticated and versatile engineering strategies, such as maIL-12 and NR4A2-driven expression (100,105,111). However, the manufacturing process required for human T-cell engineering—including leukapheresis, genetic modification, ex vivo expansion and associated quality control—together constitute a highly complex approach that may present translational challenges during clinical development (126). Moreover, this expensive therapeutic modality may not be accessible to all cancer patients, in particular owing to challenges associated with scalability and clinical delivery (127).

While both immunocytokines and ACT-based strategies enhance tumour localisation and exert potent anti-tumour effects, they require precise engineering to minimise systemic cytokine exposure and associated toxicity (128). Conversely, intratumoural administration of synthetic polymer-based depots constitutes a technically simpler approach to achieve sustained local cytokine release. Still, it lacks tumour selectivity and depends entirely on the injection site for localisation (121).

Conclusions

The pleiotropic cytokine IL-12 primarily acts through local paracrine and autocrine signalling, similar to many other cytokines (20). To prevent systemic toxicity, IL-12-based immunotherapies should be restricted to preferentially target immune cells within the TME and draining lymph nodes—such as activated T cells, NK cells, DCs, TAMs, and MDSCs—rather than circulating lymphocytes (129). Thus, concentrating biologically active IL-12 at the tumour site is crucial to achieving both safety and potent antitumour efficacy (10). Immunocytokines have the limitation that systemic administration of macromolecules—such as large fusion proteins—is challenging, given their limited ability to penetrate tumours (7). The interstitial fluid pressure (IFP) is elevated in solid tumours due to the abnormal and leaky vasculature, which acts as a physiological barrier to drug delivery (130,131). To address this issue, future strategies could include the use of nanoparticles that exploit the enhanced permeability and retention (EPR) effect or combining immunocytokines with a vasoconstrictor, such as angiotensin II (AT-II) (130,132). The hypertension induced by AT-II increases microvascular pressure within tumour tissues, thereby enhancing the convective transport and uptake of large drug molecules from blood vessels into the tumour tissues (132-134). Alternatively, ACT using tumour-specific T cells provides an opportunity to deliver IL-12 to the TME (93). Immune cells—particularly T cells, the primary effectors of tumour cell killing—often struggle to eliminate cancer due to tumour-intrinsic evasion mechanisms (135). As summarised above, a variety of genetically engineered strategies have been proposed to enable T cells to safely deliver IL-12 to the site of disease (99-101,105). These include the synthetic NFAT-IL-12 system triggered by TCR or CAR engagement, expression of membrane-bound IL-12, secretion of immunocytokines, and co-expression of chemokine receptors with IL-12. However, the risk of off-tumour, on-target toxicity—commonly observed with many adoptive cell therapies—poses a significant challenge to integrating additional immunostimulatory cytokines, as the recognition of TAAs on healthy tissues could result in unintended tissue damage (136). Advances in synthetic biology are enabling more precise control over CAR T-cell cytotoxicity, better ensuring that their activity remains confined to tumours and reducing the risk of off-tumour, on-target toxicity (136). Adjusting CAR affinity through scFv engineering has become an effective strategy to maintain sufficient sensitivity for TAA detection on malignant cells, while avoiding activation by healthy tissues with low antigen expression (137,138). Exemplifying this, in the human U87 glioma mouse xenograft model, off-tumour, on-target toxicity was shown to be highly affected by CAR affinity. Although both high and low-affinity EGFR-targeted CAR T cells were capable of inducing potent tumour regression, mice treated with high-affinity cetuximab CAR T cells developed pronounced toxicities, including marked loss of body weight and reduced activity, which led to early mortality. Conversely, mice treated with low-affinity nimotuzumab-CAR T cells displayed no detectable toxicity (139). Besides, the off-tumour toxicity mediated by CAR T cells after infusion can also be mitigated by administering the tyrosine kinase inhibitor dasatinib. This inhibitor acts as a rapid, reversible brake on CAR T-cell signalling without compromising cell functionality (140).

Despite the promising preclinical and early clinical data for the numerous novel IL-12-based treatments discussed in this review, several critical challenges remain in the effective treatment of solid tumours. While studies have shown that IL-12-driven immunotherapy reprograms the immunosuppressive milieu and improves T-cell activation and infiltration into tumours, tumour antigen heterogeneity continues to limit therapeutic efficacy (79,118). One of the key antitumour actions of IL-12 is its potentiation of T-cell-mediated cytotoxicity; therefore, its therapeutic success depends on the expression of suitable tumour antigens when combined with ACT and thus remains constrained by antigen heterogeneity (141).

The heterogeneous expression of tumour antigens in solid tumours facilitates the survival and expansion of antigen-negative clones that evade recognition by CAR-T cells. Additionally, tumours can adapt under immune pressure by downregulating or completely losing the targeted antigen, ultimately leading to relapse driven by antigen-low or antigen-deficient variants (142). To resolve this issue, one could envision combining IL-12 with nascent multispecific CARs targeting multiple antigens, which have shown promise in overcoming antigen escape and enhancing control of established tumours in preclinical models (6,143). A study employing a bispecific CAR targeting two distinct glioma-associated antigens, HER2 and IL13Rα2, demonstrated sustained and superior antitumour activity in a murine glioblastoma model, effectively mitigating antigen escape relative to mono-specific CAR T cells (targeting either HER2 or IL13Rα2 alone), thereby addressing intrapatient antigenic heterogeneity (144). Subsequently, to further circumvent antigenic variability across patients, the same research group created a trivalent CAR T-cell product targeting three glioma antigens HER2, IL13Ra2, and EphA2, which not only displayed augmented antitumour effector function, including enhanced cytolytic activity and increased cytokine secretion, but also achieved more effective elimination of established glioblastoma and prolonged survival in mice relative to its mono-specific and bi-specific CAR T-cell counterparts (145). Moreover, a phase I clinical trial published earlier this year reported that a bivalent CAR T-cell targeting the EGFR epitope 806 and IL-13Rα2 was safe and showed preliminary evidence of efficacy in patients with recurrent glioblastoma (146).

In addition, the limited translatability of toxicity findings from murine preclinical models to human clinical settings underscores the need for improved model systems that more accurately recapitulate human physiology and immunobiology (7). Many studies have been performed in immunodeficient mice, which are inadequate for modelling IL-12-induced immune-mediated adverse effects (117,118). This could be addressed by employing immunocompetent humanised tumour mouse models, in which immunodeficient hosts are engrafted with human haematopoietic stem cells, immune cells and tissues to reconstitute a functional human immune system (147). Such models would yield a more complete picture of the interfaces among the immune system, the tumour, and IL-12, supporting the design of safer and more effective IL-12 cancer therapies (148).

Non-human primates (NHPs), such as cynomolgus monkeys, rhesus macaques, squirrel monkeys and common marmosets, represent a major class of large-animal models commonly used in preclinical research (149,150). NHPs more closely match humans in their physiology and anatomy than other large-animal species, such as canines and swine, and, importantly, exhibit high immunological homology with humans, as evidenced by the extensive cross-reactivity of human immunoreagents, including cytokines and monoclonal antibodies, with NHP immune targets (149,150). In 2024, Ding and colleagues assessed the toxicological and safety profiles of repeated subcutaneous administration of recombinant human IL-12 (rhIL-12) in rhesus monkeys across a range of doses (151). Likewise, the in vivo biological responses to rhIL-12 have been evaluated in both squirrel monkeys and cynomolgus monkeys (152,153). Subsequent steps could involve evaluating IL-12 combination therapies in NHPs to address the translational gap observed with current murine models (154). However, these approaches invoke very significant ethical concerns, causing regulatory authorities to consider measures that will reduce the need for this type of research in drug development for humans (https://www.fda.gov/news-events/press-announcements/fda-releases-draft-guidance-reducing-testing-non-human-primates-monoclonal-antibodies, accessed December 14^th, 2025).

Both immunocytokines and ACT-based IL-12 platforms have enabled a variety of tumour-targeted delivery strategies. However, they may still pose a risk of systemic cytokine exposure and associated toxicities, particularly when gene circuits are leaky or when antibody fusions retain cytokine activity in the circulation (10,104). To further reduce IL-12-induced systemic toxicity, cutting-edge protein-engineering strategies include protease-sensitive masking domains that unshield IL-12 only after cleavage by tumour-associated enzymes (e.g., matrix metalloproteinases) (128,155), and the construction of structurally modified cytokines, such as split IL-12 systems in which the p35 and p40 subunits are engineered to reconstitute locally within the tumour through stepwise reassembly (10,156).

Unlike protein-engineering strategies that modify IL-12 itself, Luo et al. developed an approach that uses a nanotechnology-based delivery system in which unmodified IL-12 is encapsulated within redox-responsive human serum albumin nanoparticles and conjugated to the CAR T-cell membrane via bioorthogonal chemistry (157). IL-12 was then released from the nanoparticle in response to tumour antigen-induced increases in surface reductive thiols on activated CAR-T cells. By incorporating an IL-12 nanochaperone, this approach substantially augmented the antitumour potency of CAR T cells, while mitigating undesirable toxicity (128,157).

Successfully addressing these remaining hurdles will be pivotal to unlocking the full potential of IL-12 as a cancer immunotherapeutic (67,158). Continued innovation and careful optimisation will be essential to ensure that IL-12 can be deployed safely and help enable the next generation of cytokine-enhanced immunotherapies (125).

The proinflammatory nature of IL-12 and its ability to trigger a range of robust immune-stimulating activities suggest that it could be a valuable component of combination treatment regimens aimed at enhancing tumour immunogenicity (72). Checkpoint inhibitors perform poorly in immune-desert ‘cold’ tumours, primarily due to inadequate CD8⁺ T-cell activation. IL-12, which promotes T-cell activation and elicits immune infiltration into the TME, is an attractive candidate for synergising with checkpoint blockade (10,155). In support of this, IL-12 combined with anti-PD-1 and anti-CTLA-4 produced more durable and effective anti-tumour responses in a mouse ovarian cancer model, prolonging survival compared with IL-12 or ICI monotherapy (159). As discussed above, the work by Priceman’s group demonstrates the synergistic anti-tumour efficacy of integrating PD-L1 blockade, IL-12, and CAR T-cell therapy (114). Similarly, combining inducible membrane-anchored IL-12-engineered CAR T cells with ICIs (e.g., PD-1/PD-L1 blockade) may offer another strategy to achieve synergistic anti-tumour effects by enabling safe, tumour-restricted IL-12 delivery while counteracting the suppression imposed by the upregulation of inhibitory receptors on T cells, an underexplored approach with considerable therapeutic promise (10,114).

Acknowledgments

None.

Footnote

Peer Review File: Available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-1806/prf

Funding: None.

Conflicts of Interest: Both authors have completed the ICMJE uniform disclosure form (available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-1806/coif). J.M. is scientific founder, chief scientific officer, paid consultant and founder shareholder in Leucid Bio. The other author has no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

References

1. Wu Z, Xia F, Lin R. Global burden of cancer and associated risk factors in 204 countries and territories, 1980-2021: a systematic analysis for the GBD 2021. J Hematol Oncol 2024;17:119. [Crossref] [PubMed]
2. Bray F, Laversanne M, Sung H, et al. Global cancer statistics 2022: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin 2024;74:229-63. [Crossref] [PubMed]
3. Abbott M, Ustoyev Y. Cancer and the Immune System: The History and Background of Immunotherapy. Semin Oncol Nurs 2019;35:150923. [Crossref] [PubMed]
4. Sahu M, Suryawanshi H. Immunotherapy: The future of cancer treatment. J Oral Maxillofac Pathol 2021;25:371. [Crossref] [PubMed]
5. McCune JS. Rapid Advances in Immunotherapy to Treat Cancer. Clin Pharmacol Ther 2018;103:540-4. [Crossref] [PubMed]
6. Taylor CA, Glover M, Maher J. CAR-T cell technologies that interact with the tumour microenvironment in solid tumours. Expert Rev Clin Immunol 2024;20:849-71. [Crossref] [PubMed]
7. Geils C, Kathrein KL. Augmentation of Solid Tumor Immunotherapy With IL-12. J Gene Med 2024;26:e70000. [Crossref] [PubMed]
8. Song K. Current Development Status of Cytokines for Cancer Immunotherapy. Biomol Ther (Seoul) 2024;32:13-24. [Crossref] [PubMed]
9. Kobayashi M, Fitz L, Ryan M, et al. Identification and purification of natural killer cell stimulatory factor (NKSF), a cytokine with multiple biologic effects on human lymphocytes. J Exp Med 1989;170:827-45. [Crossref] [PubMed]
10. Nguyen KG, Vrabel MR, Mantooth SM, et al. Localized Interleukin-12 for Cancer Immunotherapy. Front Immunol 2020;11:575597. [Crossref] [PubMed]
11. Presky DH, Yang H, Minetti LJ, et al. A functional interleukin 12 receptor complex is composed of two beta-type cytokine receptor subunits. Proc Natl Acad Sci U S A 1996;93:14002-7. [Crossref] [PubMed]
12. Grohmann U, Belladonna ML, Bianchi R, et al. IL-12 acts directly on DC to promote nuclear localization of NF-kappaB and primes DC for IL-12 production. Immunity 1998;9:315-23. [Crossref] [PubMed]
13. Airoldi I, Gri G, Marshall JD, et al. Expression and function of IL-12 and IL-18 receptors on human tonsillar B cells. J Immunol 2000;165:6880-8. [Crossref] [PubMed]
14. O'Shea JJ, Gadina M, Schreiber RD. Cytokine signaling in 2002: new surprises in the Jak/Stat pathway. Cell 2002;109:S121-31. [Crossref] [PubMed]
15. Watford WT, Hissong BD, Bream JH, et al. Signaling by IL-12 and IL-23 and the immunoregulatory roles of STAT4. Immunol Rev 2004;202:139-56. [Crossref] [PubMed]
16. Watford WT, Moriguchi M, Morinobu A, et al. The biology of IL-12: coordinating innate and adaptive immune responses. Cytokine Growth Factor Rev 2003;14:361-8. [Crossref] [PubMed]
17. Bacon CM, McVicar DW, Ortaldo JR, et al. Interleukin 12 (IL-12) induces tyrosine phosphorylation of JAK2 and TYK2: differential use of Janus family tyrosine kinases by IL-2 and IL-12. J Exp Med 1995;181:399-404. [Crossref] [PubMed]
18. Naeger LK, McKinney J, Salvekar A, et al. Identification of a STAT4 binding site in the interleukin-12 receptor required for signaling. J Biol Chem 1999;274:1875-8. [Crossref] [PubMed]
19. Xu X, Sun YL, Hoey T. Cooperative DNA binding and sequence-selective recognition conferred by the STAT amino-terminal domain. Science 1996;273:794-7. [Crossref] [PubMed]
20. Ullrich KA, Schulze LL, Paap EM, et al. Immunology of IL-12: An update on functional activities and implications for disease. EXCLI J 2020;19:1563-89. [Crossref] [PubMed]
21. Liu J, Cao S, Herman LM, et al. Differential regulation of interleukin (IL)-12 p35 and p40 gene expression and interferon (IFN)-gamma-primed IL-12 production by IFN regulatory factor 1. J Exp Med 2003;198:1265-76. [Crossref] [PubMed]
22. Trinchieri G, Gerosa F. Immunoregulation by interleukin-12. J Leukoc Biol 1996;59:505-11. [Crossref] [PubMed]
23. Trinchieri G, Rengaraju M, D'Andrea A, et al. Producer cells of interleukin-12. Immunol Today 1993;14:237-8. [Crossref] [PubMed]
24. Macatonia SE, Hosken NA, Litton M, et al. Dendritic cells produce IL-12 and direct the development of Th1 cells from naive CD4+ T cells. J Immunol 1995;154:5071-9.
25. Aste-Amezaga M, D'Andrea A, Kubin M, et al. Cooperation of natural killer cell stimulatory factor/interleukin-12 with other stimuli in the induction of cytokines and cytotoxic cell-associated molecules in human T and NK cells. Cell Immunol 1994;156:480-92. [Crossref] [PubMed]
26. Valiante NM, Rengaraju M, Trinchieri G. Role of the production of natural killer cell stimulatory factor (NKSF/IL-12) in the ability of B cell lines to stimulate T and NK cell proliferation. Cell Immunol 1992;145:187-98. [Crossref] [PubMed]
27. Hsieh CS, Macatonia SE, Tripp CS, et al. Development of TH1 CD4+ T cells through IL-12 produced by Listeria-induced macrophages. Science 1993;260:547-9. [Crossref] [PubMed]
28. Trinchieri G. Interleukin-12 and the regulation of innate resistance and adaptive immunity. Nat Rev Immunol 2003;3:133-46. [Crossref] [PubMed]
29. Brunda MJ, Luistro L, Warrier RR, et al. Antitumor and antimetastatic activity of interleukin 12 against murine tumors. J Exp Med 1993;178:1223-30. [Crossref] [PubMed]
30. Kozar K, Kamiński R, Switaj T, et al. Interleukin 12-based immunotherapy improves the antitumor effectiveness of a low-dose 5-Aza-2'-deoxycitidine treatment in L1210 leukemia and B16F10 melanoma models in mice. Clin Cancer Res 2003;9:3124-33.
31. Atkins MB, Robertson MJ, Gordon M, et al. Phase I evaluation of intravenous recombinant human interleukin 12 in patients with advanced malignancies. Clin Cancer Res 1997;3:409-17.
32. Leonard JP, Sherman ML, Fisher GL, et al. Effects of single-dose interleukin-12 exposure on interleukin-12-associated toxicity and interferon-gamma production. Blood 1997;90:2541-8.
33. Motzer RJ, Rakhit A, Thompson JA, et al. Randomized multicenter phase II trial of subcutaneous recombinant human interleukin-12 versus interferon-alpha 2a for patients with advanced renal cell carcinoma. J Interferon Cytokine Res 2001;21:257-63. [Crossref] [PubMed]
34. Hurteau JA, Blessing JA, DeCesare SL, et al. Evaluation of recombinant human interleukin-12 in patients with recurrent or refractory ovarian cancer: a gynecologic oncology group study. Gynecol Oncol 2001;82:7-10. [Crossref] [PubMed]
35. Dong C, Tan D, Sun H, et al. Interleukin-12 Delivery Strategies and Advances in Tumor Immunotherapy. Curr Issues Mol Biol 2024;46:11548-79. [Crossref] [PubMed]
36. Ansell SM, Witzig TE, Kurtin PJ, et al. Phase 1 study of interleukin-12 in combination with rituximab in patients with B-cell non-Hodgkin lymphoma. Blood 2002;99:67-74. [Crossref] [PubMed]
37. Teicher BA, Ara G, Buxton D, et al. Optimal scheduling of interleukin 12 and chemotherapy in the murine MB-49 bladder carcinoma and B16 melanoma. Clin Cancer Res 1997;3:1661-7.
38. Mukhopadhyay A, Wright J, Shirley S, et al. Characterization of abscopal effects of intratumoral electroporation-mediated IL-12 gene therapy. Gene Ther 2019;26:1-15. [Crossref] [PubMed]
39. Tuomela K, Ambrose AR, Davis DM. Escaping Death: How Cancer Cells and Infected Cells Resist Cell-Mediated Cytotoxicity. Front Immunol 2022;13:867098. [Crossref] [PubMed]
40. Mirlekar B, Pylayeva-Gupta Y. IL-12 Family Cytokines in Cancer and Immunotherapy. Cancers (Basel) 2021;13:167. [Crossref] [PubMed]
41. Trinchieri G. Interleukin-12: a cytokine at the interface of inflammation and immunity. Adv Immunol 1998;70:83-243. [Crossref] [PubMed]
42. Chouaib S, Chehimi J, Bani L, et al. Interleukin 12 induces the differentiation of major histocompatibility complex class I-primed cytotoxic T-lymphocyte precursors into allospecific cytotoxic effectors. Proc Natl Acad Sci U S A 1994;91:12659-63. [Crossref] [PubMed]
43. Salcedo TW, Azzoni L, Wolf SF, et al. Modulation of perforin and granzyme messenger RNA expression in human natural killer cells. J Immunol 1993;151:2511-20.
44. Cesano A, Visonneau S, Clark SC, et al. Cellular and molecular mechanisms of activation of MHC nonrestricted cytotoxic cells by IL-12. J Immunol 1993;151:2943-57.
45. Smyth MJ, Taniguchi M, Street SE. The anti-tumor activity of IL-12: mechanisms of innate immunity that are model and dose dependent. J Immunol 2000;165:2665-70. [Crossref] [PubMed]
46. Xu Y, Sun X, Tong Y. Interleukin-12 in multimodal tumor therapies for induction of anti-tumor immunity. Discov Oncol 2024;15:170. [Crossref] [PubMed]
47. Grohmann U, Belladonna ML, Vacca C, et al. Positive regulatory role of IL-12 in macrophages and modulation by IFN-gamma. J Immunol 2001;167:221-7. [Crossref] [PubMed]
48. Murray MF. Chapter 39 - Susceptibility and Response to Infection. In: Rimoin DL, Pyeritz RE, Korf BR, editors. Emery and Rimoin's Principles and Practice of Medical Genetics (Sixth Edition). New York: Academic Press; 2013.
49. Jorgovanovic D, Song M, Wang L, et al. Roles of IFN-γ in tumor progression and regression: a review. Biomark Res 2020;8:49. [Crossref] [PubMed]
50. Steimle V, Siegrist CA, Mottet A, et al. Regulation of MHC class II expression by interferon-gamma mediated by the transactivator gene CIITA. Science 1994;265:106-9. [Crossref] [PubMed]
51. Pan J, Zhang M, Wang J, et al. Interferon-gamma is an autocrine mediator for dendritic cell maturation. Immunol Lett 2004;94:141-51. [Crossref] [PubMed]
52. Hu X, Ivashkiv LB. Cross-regulation of signaling pathways by interferon-gamma: implications for immune responses and autoimmune diseases. Immunity 2009;31:539-50. [Crossref] [PubMed]
53. Szabo SJ, Sullivan BM, Peng SL, et al. Molecular mechanisms regulating Th1 immune responses. Annu Rev Immunol 2003;21:713-58. [Crossref] [PubMed]
54. Lin Q, Rong L, Jia X, et al. IFN-γ-dependent NK cell activation is essential to metastasis suppression by engineered Salmonella. Nat Commun 2021;12:2537. [Crossref] [PubMed]
55. Bhat P, Leggatt G, Waterhouse N, et al. Interferon-γ derived from cytotoxic lymphocytes directly enhances their motility and cytotoxicity. Cell Death Dis 2017;8:e2836. [Crossref] [PubMed]
56. Song M, Ping Y, Zhang K, et al. Low-Dose IFNγ Induces Tumor Cell Stemness in Tumor Microenvironment of Non-Small Cell Lung Cancer. Cancer Res 2019;79:3737-48. [Crossref] [PubMed]
57. Ikeda H, Old LJ, Schreiber RD. The roles of IFN gamma in protection against tumor development and cancer immunoediting. Cytokine Growth Factor Rev 2002;13:95-109. [Crossref] [PubMed]
58. Kubin M, Kamoun M, Trinchieri G. Interleukin 12 synergizes with B7/CD28 interaction in inducing efficient proliferation and cytokine production of human T cells. J Exp Med 1994;180:211-22. [Crossref] [PubMed]
59. Kerkar SP, Goldszmid RS, Muranski P, et al. IL-12 triggers a programmatic change in dysfunctional myeloid-derived cells within mouse tumors. J Clin Invest 2011;121:4746-57. [Crossref] [PubMed]
60. Watkins SK, Egilmez NK, Suttles J, et al. IL-12 rapidly alters the functional profile of tumor-associated and tumor-infiltrating macrophages in vitro and in vivo. J Immunol 2007;178:1357-62. [Crossref] [PubMed]
61. Jones DS 2nd, Nardozzi JD, Sackton KL, et al. Cell surface-tethered IL-12 repolarizes the tumor immune microenvironment to enhance the efficacy of adoptive T cell therapy. Sci Adv 2022;8:eabi8075. [Crossref] [PubMed]
62. Li K, Shi H, Zhang B, et al. Myeloid-derived suppressor cells as immunosuppressive regulators and therapeutic targets in cancer. Signal Transduct Target Ther 2021;6:362. [Crossref] [PubMed]
63. Steding CE, Wu ST, Zhang Y, et al. The role of interleukin-12 on modulating myeloid-derived suppressor cells, increasing overall survival and reducing metastasis. Immunology 2011;133:221-38. [Crossref] [PubMed]
64. Vanderlugt CL, Miller SD. Epitope spreading in immune-mediated diseases: implications for immunotherapy. Nat Rev Immunol 2002;2:85-95. [Crossref] [PubMed]
65. Ma L, Hostetler A, Morgan DM, et al. Vaccine-boosted CAR T crosstalk with host immunity to reject tumors with antigen heterogeneity. Cell 2023;186:3148-3165.e20. [Crossref] [PubMed]
66. Etxeberria I, Bolaños E, Quetglas JI, et al. Intratumor Adoptive Transfer of IL-12 mRNA Transiently Engineered Antitumor CD8(+) T Cells. Cancer Cell 2019;36:613-629.e7. [Crossref] [PubMed]
67. Akorede Lawal H, Raji AO, Olayiwola AE, et al. Overcoming barriers to the implementation of interleukin-12-based strategies in cancer immunotherapy: translational challenges, clinical integration, and public health implications. Explor Med 2025;6:1001352. Available online: https://doi.org/10.37349/emed.2025.1001352
68. Lee DW, Gardner R, Porter DL, et al. Current concepts in the diagnosis and management of cytokine release syndrome. Blood 2014;124:188-95. [Crossref] [PubMed]
69. Jarczak D, Nierhaus A. Cytokine Storm-Definition, Causes, and Implications. Int J Mol Sci 2022;23:11740. [Crossref] [PubMed]
70. Jia Z, Ragoonanan D, Mahadeo KM, et al. IL12 immune therapy clinical trial review: Novel strategies for avoiding CRS-associated cytokines. Front Immunol 2022;13:952231. [Crossref] [PubMed]
71. Minnar CM, Lui G, Gulley JL, et al. Preclinical and clinical studies of a tumor targeting IL-12 immunocytokine. Front Oncol 2023;13:1321318. [Crossref] [PubMed]
72. Tugues S, Burkhard SH, Ohs I, et al. New insights into IL-12-mediated tumor suppression. Cell Death Differ 2015;22:237-46. [Crossref] [PubMed]
73. Pabani A, Gainor JF. Facts and Hopes: Immunocytokines for Cancer Immunotherapy. Clin Cancer Res 2023;29:3841-9. [Crossref] [PubMed]
74. Greiner JW, Morillon YM 2nd, Schlom J. NHS-IL12, a Tumor-Targeting Immunocytokine. Immunotargets Ther 2021;10:155-69. [Crossref] [PubMed]
75. Liu ZG, Jiao D. Necroptosis, tumor necrosis and tumorigenesis. Cell Stress 2019;4:1-8. [Crossref] [PubMed]
76. Fallon J, Tighe R, Kradjian G, et al. The immunocytokine NHS-IL12 as a potential cancer therapeutic. Oncotarget 2014;5:1869-84. [Crossref] [PubMed]
77. Schoenhaut DS, Chua AO, Wolitzky AG, et al. Cloning and expression of murine IL-12. J Immunol 1992;148:3433-40.
78. Colombo MP, Vagliani M, Spreafico F, et al. Amount of interleukin 12 available at the tumor site is critical for tumor regression. Cancer Res 1996;56:2531-4.
79. Strauss J, Heery CR, Kim JW, et al. First-in-Human Phase I Trial of a Tumor-Targeted Cytokine (NHS-IL12) in Subjects with Metastatic Solid Tumors. Clin Cancer Res 2019;25:99-109. [Crossref] [PubMed]
80. Lyerly HK, Osada T, Hartman ZC. Right Time and Place for IL12: Targeted Delivery Stimulates Immune Therapy. Clin Cancer Res 2019;25:9-11. [Crossref] [PubMed]
81. Strauss J, Deville JL, Sznol M, et al. First-in-human phase Ib trial of M9241 (NHS-IL12) plus avelumab in patients with advanced solid tumors, including dose expansion in patients with advanced urothelial carcinoma. J Immunother Cancer 2023;11:e005813. [Crossref] [PubMed]
82. Apolo AB, Ellerton JA, Infante JR, et al. Avelumab as second-line therapy for metastatic, platinum-treated urothelial carcinoma in the phase Ib JAVELIN Solid Tumor study: 2-year updated efficacy and safety analysis. J Immunother Cancer 2020;8:e001246. [Crossref] [PubMed]
83. Qian J, Wang C, Wang B, et al. The IFN-γ/PD-L1 axis between T cells and tumor microenvironment: hints for glioma anti-PD-1/PD-L1 therapy. J Neuroinflammation 2018;15:290. [Crossref] [PubMed]
84. Went P, Vasei M, Bubendorf L, et al. Frequent high-level expression of the immunotherapeutic target Ep-CAM in colon, stomach, prostate and lung cancers. Br J Cancer 2006;94:128-35. [Crossref] [PubMed]
85. Gillies SD, Lan Y, Wesolowski JS, et al. Antibody-IL-12 fusion proteins are effective in SCID mouse models of prostate and colon carcinoma metastases. J Immunol 1998;160:6195-203.
86. Ahmed M, Cheung NK. Engineering anti-GD2 monoclonal antibodies for cancer immunotherapy. FEBS Lett 2014;588:288-97. [Crossref] [PubMed]
87. Sadida HQ, Abdulla A, Marzooqi SA, et al. Epigenetic modifications: Key players in cancer heterogeneity and drug resistance. Transl Oncol 2024;39:101821. [Crossref] [PubMed]
88. Mansurov A, Ishihara J, Hosseinchi P, et al. Collagen-binding IL-12 enhances tumour inflammation and drives the complete remission of established immunologically cold mouse tumours. Nat Biomed Eng 2020;4:531-43. [Crossref] [PubMed]
89. Ishihara J, Ishihara A, Sasaki K, et al. Targeted antibody and cytokine cancer immunotherapies through collagen affinity. Sci Transl Med 2019;11:eaau3259. [Crossref] [PubMed]
90. Fabbri M, Smart C, Pardi R. T lymphocytes. Int J Biochem Cell Biol 2003;35:1004-8. [Crossref] [PubMed]
91. Karthikeyan D, Bennett SN, Reynolds AG, et al. Conditional generation of real antigen-specific T cell receptor sequences. Nat Mach Intell 2025;7:1494-509. [Crossref] [PubMed]
92. Sharpe M, Mount N. Genetically modified T cells in cancer therapy: opportunities and challenges. Dis Model Mech 2015;8:337-50. [Crossref] [PubMed]
93. Rohaan MW, Wilgenhof S, Haanen JBAG. Adoptive cellular therapies: the current landscape. Virchows Arch 2019;474:449-61. [Crossref] [PubMed]
94. Martinez M, Moon EK. CAR T Cells for Solid Tumors: New Strategies for Finding, Infiltrating, and Surviving in the Tumor Microenvironment. Front Immunol 2019;10:128. [Crossref] [PubMed]
95. Chmielewski M, Abken H. CAR T cells transform to trucks: chimeric antigen receptor-redirected T cells engineered to deliver inducible IL-12 modulate the tumour stroma to combat cancer. Cancer Immunol Immunother 2012;61:1269-77. [Crossref] [PubMed]
96. Chmielewski M, Hombach AA, Abken H. Of CARs and TRUCKs: chimeric antigen receptor (CAR) T cells engineered with an inducible cytokine to modulate the tumor stroma. Immunol Rev 2014;257:83-90. [Crossref] [PubMed]
97. Parums DV. A Review of CAR T Cells and Adoptive T-Cell Therapies in Lymphoid and Solid Organ Malignancies. Med Sci Monit 2025;31:e948125. [Crossref] [PubMed]
98. Roddie C, Sandhu KS, Tholouli E, et al. Obecabtagene Autoleucel in Adults with B-Cell Acute Lymphoblastic Leukemia. N Engl J Med 2024;391:2219-30. [Crossref] [PubMed]
99. Kerkar SP, Muranski P, Kaiser A, et al. Tumor-specific CD8+ T cells expressing interleukin-12 eradicate established cancers in lymphodepleted hosts. Cancer Res 2010;70:6725-34. [Crossref] [PubMed]
100. Zhang L, Kerkar SP, Yu Z, et al. Improving adoptive T cell therapy by targeting and controlling IL-12 expression to the tumor environment. Mol Ther 2011;19:751-9. [Crossref] [PubMed]
101. Chinnasamy D, Yu Z, Kerkar SP, et al. Local delivery of interleukin-12 using T cells targeting VEGF receptor-2 eradicates multiple vascularized tumors in mice. Clin Cancer Res 2012;18:1672-83. [Crossref] [PubMed]
102. Gaud G, Lesourne R, Love PE. Regulatory mechanisms in T cell receptor signalling. Nat Rev Immunol 2018;18:485-97. [Crossref] [PubMed]
103. Chow CW, Rincón M, Davis RJ. Requirement for transcription factor NFAT in interleukin-2 expression. Mol Cell Biol 1999;19:2300-7. [Crossref] [PubMed]
104. Zhang L, Morgan RA, Beane JD, et al. Tumor-infiltrating lymphocytes genetically engineered with an inducible gene encoding interleukin-12 for the immunotherapy of metastatic melanoma. Clin Cancer Res 2015;21:2278-88. [Crossref] [PubMed]
105. Hu J, Yang Q, Zhang W, et al. Cell membrane-anchored and tumor-targeted IL-12 (attIL12)-T cell therapy for eliminating large and heterogeneous solid tumors. J Immunother Cancer 2022;10:e003633. [Crossref] [PubMed]
106. Lee EHJ, Murad JP, Christian L, et al. Antigen-dependent IL-12 signaling in CAR T cells promotes regional to systemic disease targeting. Nat Commun 2023;14:4737. [Crossref] [PubMed]
107. Satelli A, Mitra A, Cutrera JJ, et al. Universal marker and detection tool for human sarcoma circulating tumor cells. Cancer Res 2014;74:1645-50. [Crossref] [PubMed]
108. Kueberuwa G, Kalaitsidou M, Cheadle E, et al. CD19 CAR T Cells Expressing IL-12 Eradicate Lymphoma in Fully Lymphoreplete Mice through Induction of Host Immunity. Mol Ther Oncolytics 2018;8:41-51. [Crossref] [PubMed]
109. Zhang L, Davies JS, Serna C, et al. Enhanced efficacy and limited systemic cytokine exposure with membrane-anchored interleukin-12 T-cell therapy in murine tumor models. J Immunother Cancer 2020;8:e000210. [Crossref] [PubMed]
110. Jin BY, Campbell TE, Draper LM, et al. Engineered T cells targeting E7 mediate regression of human papillomavirus cancers in a murine model. JCI Insight 2018;3:e99488. [Crossref] [PubMed]
111. Chen AXY, Yap KM, Kim JS, et al. Rewiring endogenous genes in CAR T cells for tumour-restricted payload delivery. Nature 2025;644:241-51. [Crossref] [PubMed]
112. Lai J, Mardiana S, House IG, et al. Adoptive cellular therapy with T cells expressing the dendritic cell growth factor Flt3L drives epitope spreading and antitumor immunity. Nat Immunol 2020;21:914-26. [Crossref] [PubMed]
113. Kortylewski M, Xin H, Kujawski M, et al. Regulation of the IL-23 and IL-12 balance by Stat3 signaling in the tumor microenvironment. Cancer Cell 2009;15:114-23. [Crossref] [PubMed]
114. Murad JP, Christian L, Rosa R, et al. Solid tumour CAR-T cells engineered with fusion proteins targeting PD-L1 for localized IL-12 delivery. Nat Biomed Eng 2025; Epub ahead of print. [Crossref]
115. Knopf P, Stowbur D, Hoffmann SHL, et al. Acidosis-mediated increase in IFN-γ-induced PD-L1 expression on cancer cells as an immune escape mechanism in solid tumors. Mol Cancer 2023;22:207. [Crossref] [PubMed]
116. Mengos AE, Gastineau DA, Gustafson MP. The CD14(+)HLA-DR(lo/neg) Monocyte: An Immunosuppressive Phenotype That Restrains Responses to Cancer Immunotherapy. Front Immunol 2019;10:1147. [Crossref] [PubMed]
117. Zhu Y, Wang K, Yue L, et al. Mesothelin CAR-T cells expressing tumor-targeted immunocytokine IL-12 yield durable efficacy and fewer side effects. Pharmacol Res 2024;203:107186. [Crossref] [PubMed]
118. Tian Y, Zhang L, Ping Y, et al. CCR5 and IL-12 co-expression in CAR T cells improves antitumor efficacy by reprogramming tumor microenvironment in solid tumors. Cancer Immunol Immunother 2025;74:55. [Crossref] [PubMed]
119. Liu JY, Li F, Wang LP, et al. CTL- vs Treg lymphocyte-attracting chemokines, CCL4 and CCL20, are strong reciprocal predictive markers for survival of patients with oesophageal squamous cell carcinoma. Br J Cancer 2015;113:747-55. [Crossref] [PubMed]
120. Arora A, Su G, Mathiowitz E, et al. Neoadjuvant intratumoral cytokine-loaded microspheres are superior to postoperative autologous cellular vaccines in generating systemic anti-tumor immunity. J Surg Oncol 2006;94:403-12. [Crossref] [PubMed]
121. Sabel MS, Hill H, Jong YS, et al. Neoadjuvant therapy with interleukin-12-loaded polylactic acid microspheres reduces local recurrence and distant metastases. Surgery 2001;130:470-8. [Crossref] [PubMed]
122. Egilmez NK, Jong YS, Sabel MS, et al. In situ tumor vaccination with interleukin-12-encapsulated biodegradable microspheres: induction of tumor regression and potent antitumor immunity. Cancer Res 2000;60:3832-7.
123. Egilmez NK, Jong YS, Hess SD, et al. Cytokines delivered by biodegradable microspheres promote effective suppression of human tumors by human peripheral blood lymphocytes in the SCID-Winn model. J Immunother 2000;23:190-5. [Crossref] [PubMed]
124. Tang L, Pan S, Wei X, et al. Arming CAR-T cells with cytokines and more: Innovations in the fourth-generation CAR-T development. Mol Ther 2023;31:3146-62. [Crossref] [PubMed]
125. Xue D, Hsu E, Fu YX, et al. Next-generation cytokines for cancer immunotherapy. Antib Ther 2021;4:123-33. [Crossref] [PubMed]
126. Lee NK, Chang JW. Manufacturing Cell and Gene Therapies: Challenges in Clinical Translation. Ann Lab Med 2024;44:314-23. [Crossref] [PubMed]
127. Dodson BP, Levine AD. Challenges in the translation and commercialization of cell therapies. BMC Biotechnol 2015;15:70. [Crossref] [PubMed]
128. Fu Y, Tang R, Zhao X. Engineering cytokines for cancer immunotherapy: a systematic review. Front Immunol 2023;14:1218082. [Crossref] [PubMed]
129. Hamza T, Barnett JB, Li B. Interleukin 12 a key immunoregulatory cytokine in infection applications. Int J Mol Sci 2010;11:789-806. [Crossref] [PubMed]
130. Netti PA, Baxter LT, Boucher Y, et al. Time-dependent behavior of interstitial fluid pressure in solid tumors: implications for drug delivery. Cancer Res 1995;55:5451-8.
131. Jain RK, Tong RT, Munn LL. Effect of vascular normalization by antiangiogenic therapy on interstitial hypertension, peritumor edema, and lymphatic metastasis: insights from a mathematical model. Cancer Res 2007;67:2729-35. [Crossref] [PubMed]
132. Golombek SK, May JN, Theek B, et al. Tumor targeting via EPR: Strategies to enhance patient responses. Adv Drug Deliv Rev 2018;130:17-38. [Crossref] [PubMed]
133. Li CJ, Miyamoto Y, Kojima Y, et al. Augmentation of tumour delivery of macromolecular drugs with reduced bone marrow delivery by elevating blood pressure. Br J Cancer 1993;67:975-80. [Crossref] [PubMed]
134. Hori K, Suzuki M, Saito S, et al. Changes in vessel pressure and interstitial fluid pressure of normal subcutis and subcutaneous tumor in rats due to angiotensin II. Microvasc Res 1994;48:246-56. [Crossref] [PubMed]
135. Kershaw MH, Westwood JA, Darcy PK. Gene-engineered T cells for cancer therapy. Nat Rev Cancer 2013;13:525-41. [Crossref] [PubMed]
136. Flugel CL, Majzner RG, Krenciute G, et al. Overcoming on-target, off-tumour toxicity of CAR T cell therapy for solid tumours. Nat Rev Clin Oncol 2023;20:49-62. [Crossref] [PubMed]
137. Park S, Shevlin E, Vedvyas Y, et al. Micromolar affinity CAR T cells to ICAM-1 achieves rapid tumor elimination while avoiding systemic toxicity. Sci Rep 2017;7:14366. [Crossref] [PubMed]
138. Liu X, Jiang S, Fang C, et al. Affinity-Tuned ErbB2 or EGFR Chimeric Antigen Receptor T Cells Exhibit an Increased Therapeutic Index against Tumors in Mice. Cancer Res 2015;75:3596-607. [Crossref] [PubMed]
139. Caruso HG, Hurton LV, Najjar A, et al. Tuning Sensitivity of CAR to EGFR Density Limits Recognition of Normal Tissue While Maintaining Potent Antitumor Activity. Cancer Res 2015;75:3505-18. [Crossref] [PubMed]
140. Mestermann K, Giavridis T, Weber J, et al. The tyrosine kinase inhibitor dasatinib acts as a pharmacologic on/off switch for CAR T cells. Sci Transl Med 2019;11:eaau5907. [Crossref] [PubMed]
141. Del Vecchio M, Bajetta E, Canova S, et al. Interleukin-12: biological properties and clinical application. Clin Cancer Res 2007;13:4677-85. [Crossref] [PubMed]
142. Escobar G, Berger TR, Maus MV. CAR-T cells in solid tumors: Challenges and breakthroughs. Cell Rep Med 2025;6:102353. [Crossref] [PubMed]
143. Tony LT, Stabile A, Schauer MP, et al. CAR-T Cell Therapy for Solid Tumors. Transfus Med Hemother 2025;52:96-108. Available online: https://doi.org/10.1159/000542438</unknown>
144. Hegde M, Mukherjee M, Grada Z, et al. Tandem CAR T cells targeting HER2 and IL13Rα2 mitigate tumor antigen escape. J Clin Invest 2016;126:3036-52. [Crossref] [PubMed]
145. Bielamowicz K, Fousek K, Byrd TT, et al. Trivalent CAR T cells overcome interpatient antigenic variability in glioblastoma. Neuro Oncol 2018;20:506-18. [Crossref] [PubMed]
146. Bagley SJ, Desai AS, Fraietta JA, et al. Intracerebroventricular bivalent CAR T cells targeting EGFR and IL-13Rα2 in recurrent glioblastoma: a phase 1 trial. Nat Med 2025;31:2778-87. [Crossref] [PubMed]
147. Shultz LD, Brehm MA, Garcia-Martinez JV, et al. Humanized mice for immune system investigation: progress, promise and challenges. Nat Rev Immunol 2012;12:786-98. [Crossref] [PubMed]
148. Ahmed EN, Cutmore LC, Marshall JF. Syngeneic Mouse Models for Pre-Clinical Evaluation of CAR T Cells. Cancers (Basel) 2024;16:3186. [Crossref] [PubMed]
149. Matar AJ, Crepeau RL, Duran-Struuck R. Cellular Immunotherapies in Preclinical Large Animal Models of Transplantation. Transplant Cell Ther 2021;27:36-44. [Crossref] [PubMed]
150. Han HJ, Powers SJ, Gabrielson KL. The Common Marmoset-Biomedical Research Animal Model Applications and Common Spontaneous Diseases. Toxicol Pathol 2022;50:628-37. [Crossref] [PubMed]
151. Ding H, Qin H, Wang J, et al. The repeated administration of rhIL-12 for 14 weeks in rhesus monkeys: A toxicity assessment. J Appl Toxicol 2024;44:301-12. [Crossref] [PubMed]
152. Sarmiento UM, Riley JH, Knaack PA, et al. Biologic effects of recombinant human interleukin-12 in squirrel monkeys (Sciureus saimiri). Lab Invest 1994;71:862-73.
153. Bree AG, Schlerman FJ, Kaviani MD, et al. Multiple effects on peripheral hematology following administration of recombinant human interleukin 12 to nonhuman primates. Biochem Biophys Res Commun 1994;204:1150-7. [Crossref] [PubMed]
154. Fløe LVB, Pedersen MG, Møller BK. Animal models in preclinical evaluation of CAR-T cell therapy: Advantages and limitations. Biochim Biophys Acta Rev Cancer 2025;1880:189455. [Crossref] [PubMed]
155. Mansurov A, Hosseinchi P, Chang K, et al. Masking the immunotoxicity of interleukin-12 by fusing it with a domain of its receptor via a tumour-protease-cleavable linker. Nat Biomed Eng 2022;6:819-29. [Crossref] [PubMed]
156. Venetz D, Koovely D, Weder B, et al. Targeted Reconstitution of Cytokine Activity upon Antigen Binding using Split Cytokine Antibody Fusion Proteins. J Biol Chem 2016;291:18139-47. [Crossref] [PubMed]
157. Luo Y, Chen Z, Sun M, et al. IL-12 nanochaperone-engineered CAR T cell for robust tumor-immunotherapy. Biomaterials 2022;281:121341. [Crossref] [PubMed]
158. Lu Y, Zhao F. Strategies to overcome tumour relapse caused by antigen escape after CAR T therapy. Mol Cancer 2025;24:126. [Crossref] [PubMed]
159. Pavicic PG Jr, Rayman PA, Swaidani S, et al. Immunotherapy with IL12 and PD1/CTLA4 inhibition is effective in advanced ovarian cancer and associates with reversal of myeloid cell-induced immunosuppression. Oncoimmunology 2023;12:2198185. [Crossref] [PubMed]