TAMs are often the most abundant immune cells in solid tumors and play a pivotal role in shaping the immunosuppressive landscape of the TME. Initially derived from circulating monocytes and resident macrophages, TAMs are educated by the tumor milieu to adopt an immune suppressive, pro-tumoral phenotype, becoming the population of immune suppressive macrophages (13, 14). Functionally, immunosuppressive macrophages contribute to tumor progression by secreting anti-inflammatory cytokines (IL-10, TGF-β), inhibiting cytotoxic T lymphocyte (CTL) activation, promoting regulatory T cell (Treg) expansion, and supporting angiogenesis through the production of VEGF and matrix metalloproteinases (MMPs) (15, 16). They also remodel the extracellular matrix to create a physical barrier against immune cell infiltration and facilitate metastasis by enhancing tumor cell migration. Through the expression of immune checkpoint ligands such as PD-L1 and VISTA, immunosuppressive macrophages further inhibit T cell function and contribute to immune evasion (17). Their abundance within tumors often corelate with poor prognosis and resistance to therapy (18), underscoring their importance as therapeutic targets in immunomodulation strategies.

Myeloid-derived suppressor cells (MDSCs) are immature myeloid cells of the monocyte and granulcyte lineages with strong immunosuppressive activity. In cancer, MDSCs expand significantly and accumulate in the TME under the influence of tumor-secreted factors such as CSF1, G-CSF, IL-6, and prostaglandins (19, 20). These cells suppress both innate and adaptive immunity by multiple mechanisms (21, 22). These cells inhibit T cell and NK cell cytotoxicity and block dendritic cell maturation, creating a layered barrier against effective anti-tumor immunity (17, 23). Their accumulation is associated with tumor burden, metastatic potential, and treatment resistance.

Although dendritic cells (DCs) are professional antigen-presenting cells critical for initiating adaptive immunity, their functionality is often impaired in the TME. Tumor-derived cytokines such as IL-10, VEGF, and PGE2 inhibit DC maturation, resulting in accumulation of immature or tolerogenic DCs that fail to effectively prime T cells (24, 25). These dysfunctional DCs often express low levels of MHC class II and co-stimulatory molecules, while secreting immunosuppressive mediators like TGF-β or expressing IDO, contributing to T cell anergy or tolerance rather than activation (26, 27). Consequently, the DC compartment becomes a facilitator rather than a barrier to tumor growth, and restoring DC functionality is a promising avenue for enhancing cancer immunotherapy.

Neutrophils are increasingly recognized as influential components of the TME, with tumor-associated neutrophils (TANs) demonstrating dual phenotypes: anti-tumoral (N1) or pro-tumoral (N2). The tumor context, particularly the presence of TGF-β, favors polarization toward the N2 phenotype, which promotes tumor progression through several pathways (28, 29). N2 TANs secrete proteases, ROS, and neutrophil extracellular traps (NETs) that facilitate tumor invasion and metastasis (30). They also produce chemokines such as CXCL1 and CXCL8 that attract additional immunosuppressive cells and enhance angiogenesis (31). Emerging data suggest TANs also regulate pre-metastatic niche formation, positioning them as critical players in both local tumor growth and systemic disease spread.

One of the primary ways myeloid cells contribute to immunosuppression is through the secretion of anti-inflammatory cytokines, which dampen immune activation and promote tolerance. TAMs and MDSCs secrete high levels of IL-10 TGF-β (17, 32). These cytokines inhibit the function of antigen-presenting cells (APCs), reduce MHC class II expression, and suppress the production of IL-12, which is crucial for Th1 and cytotoxic T lymphocyte responses (33, 34). TGF-β also promotes the expansion of regulatory T cells (Tregs) and blocks the cytotoxic function of CD8⁺ T cells and natural killer (NK) cells, thereby creating an immune-tolerant tumor microenvironment (TME) (34, 35). The cytokine profile skews the immune response away from anti-tumor activity and facilitates tumor progression.

Myeloid cells suppress T cell function by altering the metabolic landscape of the TME. MDSCs and TAMs express enzymes such as arginase-1 (ARG1), inducible nitric oxide synthase (iNOS), and indoleamine 2,3-dioxygenase (IDO). ARG1 depletes L-arginine, an amino acid essential for T cell proliferation and TCR ζ-chain expression (36–38). Concurrently, iNOS produces nitric oxide (NO), which impairs T cell signal transduction and can induce apoptosis (39). IDO catabolizes tryptophan into kynurenine, a metabolite known to suppress T cell proliferation and promote Treg differentiation (40). Additionally, reactive oxygen species (ROS) generated by myeloid cells cause oxidative stress and nitration of TCRs, leading to functional impairment of tumor-infiltrating lymphocytes (41, 42). These metabolic suppressive pathways represent major barriers to effective antitumor immunity and are under investigation as therapeutic targets.

Myeloid cells contribute to T cell exhaustion through the expression of immune checkpoint ligands, most notably programmed death-ligand 1 (PD-L1). Inflammatory cytokines such as IFN-γ upregulate PD-L1 on TAMs, MDSCs, and tolerogenic dendritic cells (DCs) (17, 43). PD-L1 binds to PD-1 on activated T cells, leading to reduced proliferation, impaired cytokine secretion, and eventual anergy or apoptosis (44). Beyond PD-L1, myeloid cells also express VISTA (45), Galectin-9 (ligand for TIM-3) (46), and CD155 (ligand for TIGIT) (47), which further inhibit both innate and adaptive immune responses. These checkpoint molecules contribute not only to immune suppression within the TME but also to resistance against immune checkpoint inhibitors, underscoring the importance of targeting myeloid cell-derived checkpoints in combinatorial immunotherapy strategies.

In the TME, dendritic cells (DCs) often fail to fully mature due to tumor-derived inhibitory signals such as VEGF (48), IL-10 (49), and PGE2 (50). Immature or tolerogenic DCs exhibit low expression of costimulatory molecules (CD80, CD86) and reduced MHC class I/II surface expression, rendering them ineffective at priming naïve T cells (51). Instead of initiating robust cytotoxic responses, these DCs may promote T cell anergy or contribute to Treg expansion. Some DC subsets also express IDO, adding a metabolic suppressive layer to their immunoregulatory role (52). The inability of DCs to function as effective antigen-presenting cells critically impairs the initiation and maintenance of antitumor T cell responses.

Myeloid cells influence not only cellular but also physical aspects of immunosuppression. TAMs and MDSCs secrete matrix metalloproteinases (MMPs) and other enzymes that remodel the extracellular matrix (ECM), leading to the formation of dense stromal barriers. This ECM restructuring can physically prevent the infiltration of effector T cells and NK cells into the tumor core, a hallmark of “immune-excluded” tumors (53). Additionally, these myeloid populations release vascular endothelial growth factors (VEGF) and other angiogenic factors that promote abnormal vasculature, reducing effective leukocyte trafficking and enhancing tumor perfusion (54). The combination of immune exclusion and vascular dysregulation forms a formidable barrier to immunotherapy penetration.

Myeloid cells also play an active role in maintaining a suppressive immune network by secreting chemokines that recruit other regulatory cells. For example, CCL2 recruits monocytes that differentiate into TAMs (14), CXCL12 attracts MDSCs and Tregs (55), and CCL5 enhances the recruitment of Th2 and regulatory populations (56). These feedback loops amplify the immunosuppressive milieu and ensure sustained immune evasion. Moreover, by producing IL-1β and IL-6, myeloid cells can polarize additional macrophages and neutrophils toward tumor-promoting phenotypes, further reinforcing immunosuppression within the TME (57, 58).

CSF1R, a tyrosine kinase receptor expressed primarily on monocytes, macrophages, and other myeloid lineage cells, plays a pivotal role in regulating the recruitment, survival, proliferation, and differentiation of myeloid cells (9, 59). Through paracrine and autocrine signaling, tumor cells and associated stromal elements often produce high levels of CSF1 or IL-34, activating CSF1R on circulating myeloid precursors and promoting their migration into the tumor. Once recruited, CSF1R signaling promotes the differentiation of these precursors into immunosuppressive, M2-like TAMs, which secrete anti-inflammatory cytokines, support tumor angiogenesis and metastasis, and inhibit cytotoxic T cell activity (9, 60).

CSF1R inhibitors have emerged as promising immunomodulatory agents in cancer therapy, primarily due to their ability to target TAMs and MDSCs. CSF1R inhibitors, including small-molecule tyrosine kinase inhibitors (e.g., PLX3397, BLZ945) and monoclonal antibodies targeting CSF1R or its ligands, aim to disrupt CSF/IL34-CSF1R signaling axis (9, 59, 61). In preclinical tumor models, CSF1R blockade has been shown to effectively reduce TAM numbers, reprogram TAMs toward a more inflammatory M1-like phenotype, and enhance T cell infiltration and function in glioblastoma, pancreatic ductal adenocarcinoma, breast cancer, lung cancer, melanoma, colon cancer, hepatocellular carcinoma, prostate cancer and sarcoma etc (9, 59–63).

This remodeling of the TME often results in delayed tumor progression and increased sensitivity to immune checkpoint inhibitors such as anti-PD-1/PD-L1 therapies in these cancer types (60–62). However, the response to CSF1R inhibition can vary depending on tumor type, TAM heterogeneity, and compensatory mechanisms (61–63). In preclinical models, CSF1R inhibition reduces the accumulation and immunosuppressive function of MDSCs. Blocking CSF1R signaling impairs MDSC survival and suppresses their ability to produce immunosuppressive cytokines such as IL-10 and TGF-β. This reprogramming of MDSCs leads to enhanced antigen presentation and improved T cell activation, thereby promoting antitumor immunity (62, 63).

The clinical translation of CSF1R-targeted therapies has gained substantial momentum in recent years, with numerous ongoing and completed clinical trials ( Table 1 ) (64–71). Most CSF1R inhibitors under investigation are either small-molecule tyrosine kinase inhibitors (e.g., PLX3397, BLZ945, vimseltinib) or monoclonal antibodies targeting CSF1R (64–71). Pexidartinib, the most clinically advanced CSF1R inhibitor, was approved by the FDA in 2019 for the treatment of tenosynovial giant cell tumor (TGCT) (64).

However, monotherapy with CSF1R inhibitors has shown limited efficacy in clinical trials across multiple solid tumors. One major limitation is the incomplete depletion or reprogramming of tumor-associated macrophages (TAMs), which often remain functionally immunosuppressive despite treatment (63). Furthermore, tumor heterogeneity and plasticity within the myeloid compartment can lead to compensatory recruitment of other suppressive cells, such as MDSCs or regulatory T cells, reducing the long-term impact of CSF1R blockade (9, 60, 61). In some patients, compensatory upregulation of GM-CSF or IL-34 signaling helps sustain myeloid cell populations despite CSF1R inhibition (63, 66). Clinically, on-target toxicities—such as elevated liver enzymes and cytopenias—can arise due to the disruption of tissue-resident macrophages in healthy organs (63, 68, 70).

These factors, combined with the immune-excluded nature of many tumors, often necessitate the use of CSF1R inhibitors in combination with immune checkpoint inhibitors, chemotherapy, or radiation to achieve meaningful therapeutic responses. As a result, current clinical trials are increasingly focused on combination strategies. These include combining CSF1R inhibitors with immune checkpoint inhibitors, chemotherapy, anti-angiogenic agents, or radiotherapy, aiming to synergistically overcome the immunosuppressive tumor microenvironment (63, 69).

Additionally, biomarkers of response to CSF1R inhibitors are still under development. Despite these challenges, the clinical development of CSF1R inhibitors remains a promising avenue, particularly in combination with other immunotherapies. One of the most studied indicators is the density and phenotype of tumor-associated macrophages (TAMs)—tumors with high infiltration of CD163⁺ or CD206⁺ M2-like TAMs tend to show better responses to CSF1R blockade, as these cells are highly dependent on CSF1R signaling for survival and immunosuppressive function. Elevated intratumoral or circulating levels of CSF1, the ligands for CSF1R, may also predict pathway dependency and therapeutic vulnerability (9, 63). Gene expression profiling of the tumor microenvironment can further identify macrophage-dominant signatures associated with responsiveness. Moreover, post-treatment increases in inflammatory cytokines (e.g., TNF-α, IL-6) or chemokines (e.g., CXCL9, CCL5) may reflect successful TAM reprogramming toward a pro-inflammatory state (9, 60, 61, 63).Despite these advances, no universally validated predictive biomarker exists, and the utility of CSF1, or TAM density varies by tumor type (63). Therefore, integrating multimodal biomarker strategies, such as flow cytometry, IHC, transcriptomics and single cell seq, is increasingly important to stratify patients for CSF1R-targeted therapy.

PI3Kγ is a class IB isoform of the PI3K family that plays a central role in regulating the function of myeloid cells, particularly monocytes, macrophages, and neutrophils (72, 73). PI3Kγ is predominantly expressed in leukocytes, particularly in myeloid cells, where it is activated downstream of G protein–coupled receptors (GPCRs) and receptor tyrosine kinases in response to chemokines, cytokines, and growth factors (72, 73). Activation of PI3Kγ leads to the production of phosphatidylinositol-3,4,5-trisphosphate (PIP3) at the plasma membrane, initiating a cascade of downstream signaling events that regulateintegrin activation, cytoskeletal rearrangement, chemotaxis, and immune modulation (72–76). PI3Kγ activation promotes myeloid cell recruitment to tumors, where they promote immune evasion and tumor progression (72, 75–77).

Targeting PI3Kγ has emerged as a promising strategy to reprogram TME and overcome immunosuppression driven by myeloid cells. In tumors, PI3Kγ signaling promotes the accumulation and functional polarization of TAMs and MDSCs toward immunosuppressive phenotypes, contributing to the suppression of cytotoxic T cell activity and facilitating tumor progression (72, 75–78). PI3Kγ inhibition in TAMs led to increased production of pro-inflammatory cytokines, enhanced CD8+ T cell cytotoxicity, and suppressed tumor growth in mouse models of cancers including pancreatic ductal adenocarcinoma, lung adenocarcinoma and colon cancer, attributing to the activation of NF-κB and suppression of C/EBPβ signaling pathways in TAMs (79). Further research by Kaneda and colleagues elucidated the molecular mechanisms underlying this reprogramming. They found that PI3Kγ activity in TAMs stimulates C/EBPβ-dependent expression of immunosuppressive factors like arginase and TGF-β, while suppressing NF-κB-mediated expression of pro-inflammatory cytokines. Inhibition of PI3Kγ reversed these effects, thereby restoring CD8+ T cell-dependent cytotoxicity and inhibiting tumor growth, metastasis and vascular leak (79–82). Collectively, these findings underscore the therapeutic potential of PI3Kγ inhibition in reprogramming TAMs and modulating the TME to promote immune activation and enhance anti-tumor immunity.

The clinical targeting of PI3Kγ has emerged as a promising approach to overcome myeloid-mediated immunosuppression and improve cancer immunotherapy outcomes (83–86). Eganelisib (IPI-549), a selective PI3Kγ inhibitor, has shown encouraging early results across multiple clinical settings (83–86). In the MARIO-1 Phase 1/1b trial, eganelisib demonstrated manageable toxicity and preliminary signs of clinical benefit when administered either as monotherapy or in combination with nivolumab in patients with advanced solid tumors (83, 84). Subsequent Phase II analyses in patients with advanced urothelial carcinoma (MARIO-275) indicated that eganelisib combined with nivolumab led to numerically higher objective response rates compared to nivolumab alone, suggesting enhanced immunotherapeutic synergy through modulation of the tumor microenvironment (85). Moreover, a recent study in metastatic triple-negative breast cancer (mTNBC) showed that combining eganelisib with checkpoint inhibitors and chemotherapy resulted in macrophage reprogramming toward a pro-inflammatory phenotype, increased CD8⁺ T cell infiltration, and extracellular matrix remodeling, supporting the mechanism of action observed in preclinical models (86). In summary, continued investigation in clinical trials, coupled with mechanistic insights into biomarker development, will be essential for fully realizing the clinical potential of PI3Kγ inhibitors and integrating them into next-generation immunotherapy regimens (87).

PI3Kγ inhibitors have shown strong immunomodulatory effects in preclinical models. Their translation into clinical success, like all that of other myeloid cell inhibitors, has necessitated combination with T cell targeting and/or tumor cell targeting therapies. The selection of the best combination therapies to pair with myeloid targeted theapies for cancer clinical trials can be challenging, as a diverse array of therapies can be applied to each cancer type, and multiple trials may be required to ensure clinical success. Furthermore, although predictive biomarkers such as low MHCII levels on circulating CD14+ myeloid cells and tumor PD-L1 status were evaluated in patients treated with eganelisib, patients with and without these biomarkers showed clinical responses. Currently, no definitive biomarker identifies those patients most likely to benefit from PI3Kγ-targeted therapy (83–86). Finally, PI3Kγ inhibitors, most myeloid targeted therapeutics have resulted in serum elevations in two key liver enzymes, AST and ALT, although these elevations have been transient and reversible.Together, these challenges underscore the need for continued investigation of combination approaches, refined dosing regimens, and biomarker-guided clinical development to fully realize the potential of PI3Kγ inhibitors and other myeloid targeting therapeutics in immuno-oncology.

mTOR is a central regulator of cellular metabolism, growth, and immune function, and plays a pivotal role in shaping the phenotype and function of myeloid cells in the TME (88, 89). mTOR functions through two distinct complexes: mTORC1 and mTORC2, which are activated downstream of growth factors, nutrients, and immune stimuli. In myeloid cells, mTOR signaling integrates metabolic and inflammatory cues to direct cell fate decisions, cytokine production, and immunosuppressive activity (88, 89). In macrophages, mTORC1 activation is generally associated with immune suppressive, anti-inflammatory polarization, contributing to immune suppression, angiogenesis, and tumor progression (79, 90). Similarly, in MDSCs, mTOR activity supports expansion and suppressive function by regulating glucose metabolism and promoting expression of immunosuppressive mediators (91). mTOR also governs dendritic cell maturation and function, where aberrant mTOR signaling can result in the accumulation of immature or tolerogenic DCs that fail to stimulate effective T cell responses, further weakening antitumor immunity (92).

The mTOR signaling pathway has been extensively investigated for its role in signaling downstream of tumor cell oncogenes such as PIK3CA and KRAS. Inhibitors of mTOR were developed as inhibitors of tumor cell proliferation and survival. However, the inhibition of the mTOR signaling pathway has emerged as a promising strategy to modulate the function of immunosuppressive myeloid cells in the TME. mTOR inhibitors, including rapamycin (sirolimus) and its analogs (everolimus, temsirolimus), can disrupt these processes and reprogram the immunosuppressive function of TAMs and MDSCs (93, 94). In preclinical tumor models, mTOR inhibition has been shown to reduce the accumulation of MDSCs in tumors and to promote the differentiation of TAMs into M1-like, inflammatory macrophages capable of enhancing antigen presentation and stimulating T cell activation in hepatocellular carcinoma, breast cancer, lung cancer, colorectal cancer, melanoma, ovarian cancer and pancreatic cancer (93, 94). Importantly, mTOR inhibitors have demonstrated synergistic potential when combined with immune checkpoint blockade therapies by reducing TAM and MDSC-mediated suppression (94). However, the use of mTOR inhibitors requires careful dosing and timing, as mTOR is also critical for the proliferation and function of effector T cells (95).

The clinical development of mTOR inhibitors for cancer therapy has significantly advanced over the past two decades (96–105). The most established mTOR inhibitors are rapamycin (sirolimus) and its analogs, which include everolimus, temsirolimus, and ridaforolimus (96–100). The clinical benefits of first-generation mTOR inhibitors have been limited by modest overall response rates, cytostatic rather than cytotoxic activity, and the development of adaptive resistance mechanisms. To address this, second-generation mTOR inhibitors—such as dual mTORC1/mTORC2 inhibitors (e.g., sapanisertib, vistusertib) and dual PI3K/mTOR inhibitors (e.g., dactolisib, apitolisib)—have been developed and are undergoing evaluation in early-phase clinical trials (101–105).

While preclinical data suggest these therapeutics also alter myeloid cell properties in tumors, it is unclear whether these drugs affected myeloid cells in these clinical studies. Although these mTOR inhibitors have shown clinical efficacy in select cancers, their broader application as myeloid compartment therapeutics is limited. Feedback activation of upstream pathways, especially AKT and PI3K, due to mTORC1 inhibition, can undermine therapeutic efficacy and promote resistance (106). mTOR inhibitors can also dampen antitumor T cell responses and thus complicate their use in combination with immunotherapies (106, 107). Toxicities such as stomatitis, hyperlipidemia, hyperglycemia, and non-infectious pneumonitis limit dose intensity and long-term administration in some patients (96–102). The clinical benefits of mTOR inhibitors have been limited by modest overall response rates, and the development of adaptive resistance mechanisms (106–108). It is possible that these drugs target myeloid cells as well as tumor cells and could be used in limited doses in combination with checkpoint inhibitors and other therapeutics.Preclinical studies have demonstrated that transient or combinatorial mTOR inhibition can enhance the efficacy of immune checkpoint blockade therapies, and several combination regimens are being evaluated clinically96,97.

Predictive biomarkers for mTOR inhibitor response remain an area of active investigation, given the variable efficacy of these agents across different tumor types. Recent studies have also suggested that baseline immune context—levels of myeloid-derived suppressor cells (MDSCs) or T cell exhaustion markers—may influence responsiveness, especially when mTOR inhibitors are combined with immunotherapy (88, 92, 93). However, no single biomarker has been validated across cancer types, and heterogeneity within the tumor and the tumor microenvironment may limit the predictive power of biomarker of response. Consequently, a composite biomarker strategy incorporating genomic, proteomic, and immune parameters may be required to accurately identify patients who are most likely to benefit from mTOR-targeted therapy (106–108). Ongoing studies are focused on identifying further biomarkers of response, refining patient selection, and determining optimal dosing strategies that balance anti-tumor efficacy with preservation of immune function (108).

Syk is a non-receptor tyrosine kinase that plays a pivotal role in myeloid cell activation, functioning as a central mediator in immune receptor signaling (109, 110). Expressed in various innate immune cells, Syk is primarily activated downstream of immunoreceptors (110). Upon ligand engagement, Syk is recruited to phosphorylated ITAM motifs, where it initiates intracellular signaling cascades that regulate cytokine production, phagocytosis, oxidative burst, cellular adhesion, and antigen presentation (109–111). In macrophages and dendritic cells, Syk activation contributes to the induction of pro-inflammatory cytokines, which are critical for orchestrating innate immune responses and bridging to adaptive immunity (100, 101). Syk also modulates antigen processing and presentation (109, 110).

Therapeutic targeting of Syk has emerged as a novel strategy for reprogramming the TME by modulating the immunosuppressive functions of myeloid cells, particularly TAMs and MDSCs (111–113). In TAMs, Syk activation supports an M2-like, immunosuppressive profile, suppression of T cell activity, and promotion of angiogenesis and extracellular matrix remodeling (113). Syk also facilitates the crosstalk between TAMs and other suppressive cells, including Tregs and MDSCs, contributing to an immune-excluded or immune-silent TME in breast cancer, pancreatic ductal adenocarcinoma, colorectal cancer, ovarian cancer, lung cancer and melanoma (111–114).

Inhibition of Syk has been shown to disrupt these signaling circuits. For example, in models of chronic lymphocytic leukemia (CLL), the Syk inhibitor fostamatinib (R788) reduced tumor cell survival and disrupted B cell receptor (BCR) signaling, which is crucial for leukemic cell maintenance (115, 116). Syk inhibition also impaired the expansion and suppressive activity of myeloid-derived suppressor cells (MDSCs) in certain settings, suggesting broad potential for modulating the TME (112–115).

Clinically, several Syk inhibtors had been in clinical trials ( Table 2 ) (115–120). Fostamatinib, an oral Syk inhibitor developed for immune thrombocytopenia, has been repurposed for oncology. In early-phase clinical trials involving patients with hematologic malignancies, including CLL and diffuse large B-cell lymphoma (DLBCL), fostamatinib showed evidence of disease stabilization and partial responses, particularly in heavily pretreated patients (115). Another Syk inhibitor, entospletinib (GS-9973), was developed as a second-generation agent with improved selectivity and pharmacokinetics (116, 117). In the solid tumor setting, the application of Syk inhibitors is still in the exploratory phase (116–120).

Despite promising preclinical data, the clinical development of Syk inhibitors has been limited by several challenges. One major issue is the pleiotropic role of Syk in different cell types, which will raise concerns about off-target immunosuppression or unexpected immune dysregulation when systemically inhibited (121). Additionally, single-agent efficacy of Syk inhibitors in solid tumors has been modest, possibly due to the redundancy of downstream pathways and compensatory signaling (114, 115). Pharmacokinetically, early Syk inhibitors such as fostamatinib have shown limited selectivity and off-target effects, leading to toxicity profiles including cytopenias, hypertension, and gastrointestinal adverse events (115). Finally, the lack of validated biomarkers for patient stratification or pharmacodynamic monitoring makes it difficult to identify responsive patients and optimize treatment regimens in clinical trials. Ongoing research is focused on identifying predictive biomarkers, optimizing dosing regimens, and developing more selective Syk inhibitors to maximize efficacy while minimizing systemic toxicity in clinical trails (121).

MerTK and Axl are receptor tyrosine kinases that play critical roles in the regulation of innate immunity, particularly in the polarization of myeloid cells such as macrophages, DCs, and MDSCs. These receptors are activated by ligands such as GAS6 and Protein S, often in complex with phosphatidylserine (PtdSer) exposed on apoptotic cells (122). Upon ligand engagement, MerTK and Axl signaling cascades that promote anti-inflammatory responses, phagocytosis of apoptotic cells (efferocytosis), and the maintenance of immune tolerance (123). In the TME, MerTK and Axl are frequently upregulated in TAMs, where they promote an M2-like immunosuppressive phenotype. This polarization is leading to suppression of CD8⁺ T cell activity and facilitation of tumor growth, angiogenesis, and tissue remodeling (122, 123). Additionally, Axl signaling in dendritic cells inhibits maturation and antigen presentation, further weakening adaptive immune responses (124, 125). As such, MerTK and Axl are central to establishing and sustaining a tolerogenic, tumor-permissive environment.

MerTK and Axl have become promising targets for cancer immunotherapy, particularly in tumors with a high burden of myeloid-driven immune suppression. Several inhibition strategies have been developed, including small-molecule tyrosine kinase inhibitors (TKIs), monoclonal antibodies, and decoy receptors that interfere with ligand binding.Selective TKIs such as bemcentinib (BGB324) for Axl (126) and MRX-2843 for MerTK/FLT3 dual inhibition (127) have shown potential in blocking immune suppressive macrophage polarization, restoring antigen presentation, and enhancing pro-inflammatory cytokine production. These effects lead to increased T cell infiltration and anti-tumor immune responses (126–128).

In preclinical studies, bemcentinib (BGB324), a selective Axl inhibitor, has demonstrated the ability to block immunosuppressive macrophage polarization and restore antigen presentation in models of non-small cell lung cancer (NSCLC), triple-negative breast cancer (TNBC), melanoma, and pancreatic ductal adenocarcinoma (PDAC). These effects led to enhanced pro-inflammatory cytokine production and T cell infiltration, contributing to more effective anti-tumor immune responses (126). Similarly, MRX-2843, a dual MerTK/Axl inhibitor, has shown immune-modulatory activity in acute myeloid leukemia (AML), lung adenocarcinoma, ovarian cancer, and pediatric gliomas, where it reprogrammed tumor-associated macrophages and improved T cell function (127). These findings highlight the therapeutic potential of TAM receptor blockade across both solid tumors and hematologic malignancies.

Importantly, MerTK/Axl inhibition also reduces the accumulation and suppressive function of MDSCs, further relieving immune suppression (128). Combination therapies are actively being explored, pairing MerTK/Axl inhibitors with checkpoint inhibitors, chemotherapy, or radiation therapy to synergistically boost immune responses and overcome resistance to monotherapies (125, 129, 130). For example, in pancreatic cancer and breast cancer models, combining Axl inhibition (e.g., bemcentinib) with radiation therapy or chemotherapy (such as paclitaxel or gemcitabine) reprograms tumor-associated macrophages (TAMs), increases antigen presentation, and improves CD8⁺ T cell infiltration, leading to more robust immune responses (125). In non-small cell lung cancer (NSCLC) and melanoma, Axl inhibitors have been paired with checkpoint blockade therapies (e.g., anti–PD-1 or anti–PD-L1), which can convert immune-excluded tumors into inflamed, T cell–infiltrated environments (125). Similarly, MRX-2843, a dual MerTK/Axl inhibitor, has shown preclinical synergy with checkpoint inhibitors in AML, gliomas, and ovarian cancer by blocking myeloid-driven immune suppression (130). These combination strategies aim to neutralize immunosuppressive cues from the tumor microenvironment and restore effective anti-tumor immunity that is often limited with monotherapy approaches.

In patient studies, elevated expression of MerTK and Axl in TAMs or circulating monocytes has been correlated with poor prognosis, advanced disease stage, and resistance to immunotherapy in cancers (131–135). Clinical trials of bemcentinib (BGB324), a selective Axl tyrosine kinase inhibitor, have demonstrated promising activity in a range of Axl-expressing cancers, both as monotherapy and in combination with immune checkpoint inhibitors. In a Phase II trial (NCT03184571), bemcentinib combined with pembrolizumab showed signs of clinical benefit in Axl-high non-small cell lung cancer (NSCLC), particularly in patients with limited response to prior therapies. Similarly, in mesothelioma, the combination of bemcentinib and durvalumab (anti–PD-L1) improved immune infiltration and disease stabilization in patients who had previously progressed on standard treatment. Monotherapy studies have also shown that bemcentinib is well tolerated and has disease-modulating effects in acute myeloid leukemia (AML) and triple-negative breast cancer (TNBC), especially in tumors exhibiting epithelial–mesenchymal transition (EMT) phenotypes driven by Axl. These studies support the rationale for targeting Axl to sensitize tumors to immunotherapy and overcome resistance mechanisms associated with immune exclusion and myeloid-derived suppression (131–134). MRX-2843 is a first-in-class, orally available small-molecule dual inhibitor of MerTK and FLT3 that is currently under clinical investigation for both hematologic malignancies and solid tumors. In a Phase I clinical trial (NCT04848116), MRX-2843 has shown a favorable safety profile and early signs of anti-tumor activity in patients with relapsed or refractory acute myeloid leukemia (AML) and T-cell acute lymphoblastic leukemia (T-ALL), where aberrant MerTK and FLT3 signaling drive disease progression and therapy resistance. Additionally, MRX-2843 has been granted Orphan Drug Designation for AML and T-ALL by the FDA. The agent is also being explored in early-phase trials for pediatric gliomas, where MerTK is implicated in immune evasion and tumor proliferation. In these studies, MRX-2843 not only directly targets leukemic cells but also reprograms the tumor microenvironment by suppressing immunosuppressive macrophage signaling, offering a dual mechanism of action through both tumor-intrinsic and immune-mediated pathways (127, 128, 130, 135).Together, these findings underscore the importance of MerTK and Axl in myeloid-mediated immune tolerance and provide a strong rationale for their continued development as therapeutic targets to reprogram the TME and improve cancer immunotherapy outcomes.

Despite compelling preclinical evidence showing that MerTK and Axl inhibitors can suppress immunosuppressive macrophage polarization, enhance antigen presentation, and improve response to checkpoint inhibitors, their clinical development faces multiple challenges. One major issue is the broad expression of TAM receptors (Tyro3, Axl, MerTK) across both tumor and normal tissues, which increases the risk of off-tumor effects such as impaired clearance of apoptotic cells and autoimmune-like toxicities. Another challenge is functional redundancy among TAM receptors. Inhibiting Axl or MerTK alone may be insufficient due to compensatory upregulation of other family members, potentially limiting therapeutic efficacy (136–138). Clinically, limited biomarker availability hinders patient stratification. While Axl expression correlates with poor prognosis in many cancers, predictive biomarkers for inhibitor sensitivity remain undefined. Additionally, toxicity profiles, including thrombocytopenia, hepatic enzyme elevation, and fatigue, have been reported in early-phase trials, necessitating dose reductions that may compromise efficacy (132–134). Finally, the heterogeneity of the tumor microenvironment (TME) may influence response to MerTK/Axl inhibition, emphasizing the need for rational combination strategies and robust pharmacodynamic markers in future clinical development (136–138).

Triggering Receptor Expressed on Myeloid Cells 2 (TREM2) is a transmembrane receptor predominantly expressed on myeloid cells, including macrophages, dendritic cells, and microglia that recognizes phospholipids and triggers phagocytosis of apoptotic cells (139). In the TME, unlike classical pattern recognition receptors (e.g., TLRs, CSF1R, CD40) that primarily trigger immune activation, inflammation, or differentiation, TREM2 mainly promotes cell survival, lipid metabolism, and anti-inflammatory tissue-remodeling functions. TREM2 is highly expressed on a subset of TAMs that exhibit immunosuppressive, tissue-remodeling, and pro-tumorigenic properties (140). Unlike other receptors, upon engagement with endogenous ligands—such as phospholipids, apoptotic cells, and lipoproteins—TREM2 signals through the adaptor protein DAP12, leading to phosphorylation of its ITAM motif and activation of downstream pathways including PI3K-AKT and ERK, thereby promoting cell survival, proliferation, and anti-inflammatory cytokine production (139–141). In the tumor microenvironment (TME), TREM2+ macrophages display a transcriptional program enriched for immunosuppressive mediators such as IL-10, TGF-β, and arginase-1 (ARG1), and show reduced expression of antigen-presentation molecules (e.g., MHC-II) (142, 143). Functionally, these TREM2+ tumor-associated macrophages (TAMs) inhibit cytotoxic T lymphocyte (CTL) recruitment and activation, facilitate extracellular matrix (ECM) remodeling, and promote an immune-excluded tumor architecture. Moreover, TREM2 signaling facilitates immune evasion by creating a suppressive niche that limits dendritic cell maturation and T cell recruitment (143–145). TREM2 signaling also suppresses the production of inflammatory cytokines like IL-12 and TNF-α, which are critical for priming effective T cell responses (140, 141). In summary, TREM2⁺ TAMs are immunosuppressive, and TREM2 blockade restores anti-tumor immunity (139).

Targeting TREM2 presents a novel immunotherapeutic strategy aimed at reprogramming the myeloid compartment and enhancing anti-tumor immune responses. Inhibiting TREM2 signaling can disrupt the immunosuppressive functions of TREM2⁺ TAMs and convert them into a more pro-inflammatory, antigen-presenting phenotype (139). Several approaches have been developed to achieve this, including ( Figure 4 ): 1). Monoclonal antibodies that block TREM2 ligand binding or induce antibody-dependent depletion of TREM2⁺ TAMs (145); 2). Genetic ablation or CRISPR-mediated knockout of TREM2 in preclinical models to assess the effects on macrophage function and tumor immunity (146); 3). Small-molecule inhibitors or decoy receptors to prevent TREM2-DAP12 signaling activation (145, 146). These strategies aim to reduce TAM-mediated suppression, increase CD8⁺ T cell infiltration, and improve the efficacy of checkpoint inhibitors.Importantly, combination strategies—such as TREM2 blockade plus anti–PD-1/PD-L1 therapy—are being actively explored due to their synergistic effects in reversing T cell exclusion and enhancing tumor clearance (147–149).

Preclinical studies across various cancer types suggest that TREM2 is a promising therapeutic target for cancer immunotherapy.In non-small cell lung cancer (NSCLC), TREM2⁺ TAMs are enriched in immunologically “cold” tumors and correlate with poor response to anti–PD-1 therapy. Inhibiting TREM2 signaling reprograms these cells toward a pro-inflammatory phenotype, increasing MHC-II and co-stimulatory molecule expression, which restores CD8⁺ T cell infiltration and improves responsiveness to immune checkpoint blockade (140, 142). In hepatocellular carcinoma (HCC), TREM2 is highly expressed on myeloid cells in the tumor microenvironment and drives an immunosuppressive program. Genetic deletion or antibody-mediated blockade of TREM2 in HCC models leads to enhanced IL-12 production and antigen presentation capacity, facilitating stronger T cell activation and reduced tumor growth (145, 147). In colorectal cancer (CRC), TREM2⁺ TAMs contribute to immune evasion through suppression of dendritic cell recruitment and cytokine production. TREM2 knockout in murine CRC models results in a shift toward an M1-like TAM phenotype, marked by increased TNF-α and IL-1β, and restores local cytotoxic T cell activity (146). In pancreatic ductal adenocarcinoma (PDAC), a tumor type typically resistant to immunotherapy, TREM2 expression defines a distinct subset of TAMs that limit antigen presentation and T cell entry. Blocking TREM2 signaling promotes cross-presentation of tumor antigens, boosts CD80/CD86 levels, and synergizes with anti–PD-1 therapy to control tumor growth (142, 143). In triple-negative breast cancer (TNBC) and other aggressive breast cancers, TREM2⁺ macrophages accumulate within the tumor stroma and suppress effective immune priming. Antibody-mediated TREM2 inhibition reprograms these macrophages into a pro-inflammatory, T cell–recruiting phenotype, enhancing therapeutic responses to immunotherapy. In melanoma, single-cell transcriptomic analyses reveal that TREM2 is upregulated on immunosuppressive TAMs associated with immune checkpoint resistance. TREM2 blockade in murine melanoma models results in enhanced IFN-γ signaling, macrophage repolarization, and a more permissive environment for T cell–mediated tumor clearance. In glioblastoma, where the microenvironment is heavily myeloid-dominated, TREM2 is expressed on both TAMs and microglia, promoting tumor-supportive inflammation. Genetic deletion of TREM2 delays glioma progression, partly through increased antigen presentation and recruitment of cytotoxic lymphocytes (150–152).

Encouraged by these findings, early-phase clinical trials are underway to evaluate TREM2-targeted agents in humans. One of the leading therapeutic candidates is PY314, a humanized anti-TREM2 monoclonal antibody developed by Pionyr Immunotherapeutics, which is currently being evaluated in a Phase I clinical trial (NCT04691375) in patients with advanced solid tumors, including non-small cell lung cancer (NSCLC), renal cell carcinoma, and colorectal cancer. This trial is evaluating the safety, pharmacokinetics, and preliminary efficacy of PY314 both as monotherapy and in combination with nivolumab, an anti–PD-1 checkpoint inhibitor (153–155).

Although TREM2 inhibition has emerged as a promising strategy, its clinical application faces several key challenges. TREM2⁺ macrophages are often enriched in immune-excluded or “cold” tumors, but their distribution and functional role vary greatly across cancer types and patient populations, complicating patient selection and trial design clinically. Furthermore, TREM2 plays a dual role in tissue homeostasis and inflammation. While blocking TREM2 may enhance antigen presentation and T cell infiltration, it also carries the risk of overactivating myeloid cells, potentially leading to uncontrolled inflammation or autoimmune-like adverse events, particularly in organs with a high density of tissue-resident macrophages. From a pharmacological standpoint, the development of TREM2-targeted agents is still in early clinical stages. Most data are preclinical, and human safety and efficacy profiles remain largely undefined. Additionally, TREM2 lacks robust circulating biomarkers, making it difficult to monitor target engagement or stratify responders in ongoing trials. In some models, TREM2⁺ macrophages may coexist with other immunosuppressive populations, suggesting that TREM2 inhibition alone may not fully remodel the tumor microenvironment, thus requiring combination therapies. Lastly, heterogeneity in TREM2 gene expression and mutations raises the possibility of off-tumor risks or long-term effects on central immune regulation, necessitating careful patient screening and extended follow-up in clinical settings (143, 145, 150, 153, 155). Future directions for TREM2 inhibition include identifying predictive biomarkers for response, optimizing combination regimens, and expanding indications to other immunologically resistant cancers (155).

The Leukocyte Immunoglobulin-like Receptor Subfamily B (LILRB) family comprises inhibitory receptors predominantly expressed on myeloid cells such as macrophages, monocytes, dendritic cells, and certain subsets of granulocytes (156). Members of this family, including LILRB1–LILRB5, contain immunoreceptor tyrosine-based inhibitory motifs (ITIMs) in their cytoplasmic domains, which, upon ligand binding, recruit phosphatases such as SHP-1 and SHP-2 to suppress activating signaling pathways (156, 157). In the TME, LILRBs are often co-opted to maintain an immunosuppressive myeloid phenotype, particularly in TAMs (158) and MDSCs (159). Engagement of LILRBs by ligands such as MHC class I molecules, leads to the inhibition of myeloid activation, decreased pro-inflammatory cytokine secretion, and impaired antigen presentation (159, 160).

In non-small cell lung cancer (NSCLC), LILRB2 is highly expressed on tumor-associated macrophages (TAMs) and dendritic cells, contributing to immune suppression and poor antigen presentation. Preclinical models show that LILRB2 blockade reprograms myeloid cells to a pro-inflammatory phenotype, enhances MHC-II and CD86 expression, and synergizes with anti–PD-1 therapy to improve T cell infiltration and tumor regression (157, 158). In colorectal cancer (CRC), dual inhibition of LILRB1 and PD-L1 leads to enhanced dendritic cell activation, improved cross-presentation of tumor antigens, and greater expansion of tumor-specific CD8⁺ T cells, outperforming either monotherapy alone (157, 161). In acute myeloid leukemia (AML), LILRB2 is upregulated on leukemic stem cells and immunosuppressive myeloid cells. Blocking LILRB2, especially in conjunction with cytokine-based therapies like IL-15 superagonists, facilitates the clearance of leukemic cells by restoring innate immune activation and promoting NK and T cell responses (159). In hepatocellular carcinoma (HCC), LILRB1/LILRB2 suppress cytokine production by liver-infiltrating myeloid cells. Inhibition of these receptors, especially when combined with tumor vaccines or anti–PD-1, has been shown to increase IFN-γ and IL-12 production, supporting a pro-inflammatory tumor microenvironment (157, 161, 162). In melanoma, LILRB1 is enriched on tumor-infiltrating MDSCs and TAMs. Blocking LILRB1 signaling in combination with checkpoint blockade enhances cytotoxic T cell infiltration, remodels the tumor immune landscape, and delays tumor growth in preclinical models (163, 164).

In summary, targeting LILRBs, particularly LILRB1 and LILRB2,may help reprogram suppressive myeloid cells into a pro-inflammatory phenotype, enhance antigen presentation, and support the recruitment and activation of effector T cells. Inhibition of LILRB signaling may also synergize with anti–PD-1/PD-L1 therapies, tumor vaccines, or cytokine-based treatments to overcome immune resistance and broaden the efficacy of immunotherapy (157, 161, 163–165).

LILRB-targeted agents are advancing into clinical development based on strong preclinical evidence supporting their role in immune suppression and resistance to checkpoint inhibitors. Among the most advanced clinical-stage programs is IO-202, a humanized monoclonal antibody developed by Immune-Onc Therapeutics, which targets LILRB4. It is currently being tested in a Phase I clinical trial (NCT04372433) for acute myeloid leukemia (AML) and chronic myelomonocytic leukemia (CMML), both as monotherapy and in combination with nivolumab. Early data suggest that IO-202 is well tolerated and shows signs of immune activation, including enhanced T cell infiltration and pro-inflammatory cytokine profiles (162, 163). Another agent, NG-641, an oncolytic viral vector encoding an anti–LILRB2 antibody, is in early-phase clinical trials for patients with solid tumors, including NSCLC and colorectal cancer. This vector also expresses chemokines and immune-stimulatory molecules, aiming to remodel the tumor microenvironment and overcome resistance to PD-1 blockade. These trials represent a growing effort to leverage LILRB inhibition not only to directly relieve myeloid immunosuppression, but also to synergize with existing immunotherapies in cancers where monotherapy checkpoint inhibition has shown limited success (164, 165).

Several clinical challenges hinder the progress of LILRB-targeted therapies. One major obstacle is the broad expression and structural similarity of LILRB family members. Since LILRBs are involved in maintaining self-tolerance and immune homeostasis, their blockade may induce autoimmune or inflammatory toxicities, especially when combined with other immunotherapies such as PD-1 or CTLA-4 inhibitors. Another issue is the lack of robust predictive biomarkers to stratify patients likely to benefit from LILRB-targeted agents. As research progresses, biomarkers of LILRB expression, such as tumor-associated MHC-I/HLA-G levels or myeloid cell signatures, may help stratify patients most likely to benefit from LILRB-targeted therapy. However, More biomarkers for LILRB-targeted therapy are needed clinically. While LILRB1/2 overexpression has been observed in some cancers including AML, prostate, liver, and breast cancer, there is limited information on the functional heterogeneity of LILRB-expressing cells in the tumor microenvironment across patients. This heterogeneity contributes to variable therapeutic responses and complicates clinical trial design. Additionally, preclinical models of LILRB biology are limited, especially since LILRB signaling differs significantly between mice and humans. This translational gap poses difficulties for dosing, efficacy prediction, and safety assessment in early-phase trials. Lastly, early clinical data are sparse, and most compounds remain in preclinical or Phase I trials, with few published results on efficacy or toxicity, further delaying clinical adoption (162–165).

VISTA is a unique immune checkpoint molecule predominantly expressed on myeloid cells (166). VISTA binds to VSIG3 and PSGL-1 and regulates immune homeostasis, particularly under inflammatory or tumorigenic conditions (167). VISTA functions through mechanisms that suppress T cell proliferation, cytokine production, and antigen-specific activation, thereby contributing to immune tolerance (166, 167). In the TME, VISTA is highly expressed on immunosuppressive myeloid populations and also on T cells It exerts its effects by engaging receptors on T cells and other myeloid cells, delivering inhibitory signals that blunt effector function (167). Moreover, VISTA upregulation is often observed in tumors resistant to PD-1/PD-L1 blockade, suggesting it plays a compensatory role in maintaining immune suppression. This makes VISTA a critical checkpoint for regulating myeloid–T cell interactions and a promising target in T cell–excluded tumors (167, 168).

Therapeutic strategies aimed at blocking VISTA signaling seek to reprogram the suppressive myeloid compartment and restore T cell function within the TME. These approaches include (1):. Monoclonal antibodies that block VISTA’s interaction with its ligands or receptors, thereby lifting the suppressive signals on T cells (169) (2);. Bispecific antibodies targeting VISTA alongside other checkpoints to simultaneously engage multiple arms of immune suppression (170) (3);. Antagonistic antibodies that bind VISTA on antigen-presenting cells to reprogram them toward a pro-inflammatory phenotype, boosting antigen presentation and co-stimulation (171).

In non-small cell lung cancer (NSCLC), VISTA is highly expressed on myeloid-derived suppressor cells (MDSCs) and regulatory T cells (Tregs) within the tumor microenvironment (TME), contributing to immune suppression and resistance to PD-1 blockade. Preclinical studies demonstrate that VISTA inhibition significantly increases CD8⁺ T cell infiltration, enhances IFN-γ and TNF-α secretion, and restores anti-tumor immunity, particularly when combined with anti–PD-1 therapy in PD-1-resistant models (172). In melanoma, VISTA expression rises following PD-1/CTLA-4 blockade, suggesting a compensatory upregulation. Combining VISTA inhibitors with anti–CTLA-4 or anti–PD-1 antibodies overcomes adaptive resistance mechanisms and leads to robust tumor regression in murine melanoma models by reducing Treg frequencies and increasing granzyme B+ cytotoxic T lymphocytes in the tumor (171, 173). In pancreatic ductal adenocarcinoma (PDAC), an immunologically “cold” tumor, VISTA is highly expressed on infiltrating macrophages and MDSCs. VISTA blockade reprograms macrophages to an M1-like phenotype and allows for immune priming, which synergizes with PD-L1 inhibition to promote T cell–mediated tumor control in preclinical PDAC models (169, 170, 173). In prostate cancer, VISTA expression on TAMs correlates with poor T cell infiltration and immune exclusion. Targeting VISTA, especially in combination with cytokine-based treatments (e.g., IL-12 or IFN-α), reduces suppressive myeloid cell activity and enhances responsiveness to checkpoint inhibitors (174). In glioblastoma, VISTA is enriched in myeloid cells and contributes to the highly immunosuppressive TME. Preclinical evidence suggests that VISTA blockade improves antigen presentation and T cell infiltration and works synergistically with PD-1 blockade to overcome resistance in this typically refractory cancer (169, 170, 173).

In summary, Blocking VISTA has been shown in preclinical models to enhance CD8⁺ T cell infiltration, increase pro-inflammatory cytokine production, and reduce Treg and MDSC activity (173). Importantly, VISTA inhibition can work synergistically with PD-1/PD-L1 or CTLA-4 blockade, overcoming resistance mechanisms in immunotherapy-refractory tumors (169, 173).

The promising preclinical data supporting VISTA as a myeloid checkpoint has led to the advancement of several anti-VISTA therapies into clinical trials. Among the leading candidates is CA-170, an oral small-molecule antagonist targeting both VISTA and PD-L1, developed by Curis, Inc. CA-170 entered phase I clinical trials in patients with advanced solid tumors and lymphomas and showed a favorable safety profile, though efficacy data remains preliminary (173). Another notable agent is JNJ-61610588 (CI-8993), a humanized monoclonal antibody against VISTA developed by Janssen, currently being tested in early-phase clinical trials (e.g., NCT04475523) for advanced cancers (172, 174, 175).

While anti-VISTA therapies are still in the early clinical development stage, they hold great promise, especially in tumors with high myeloid infiltration and resistance to traditional checkpoint inhibitors (170–173, 176, 177). However, its clinical translation still faces several key challenges. One major issue is context-dependent expression. VISTA is predominantly expressed on myeloid cells and naïve T cells, and its expression can dynamically shift depending on tumor type, stage, and microenvironmental cues. This makes patient selection and biomarker development particularly difficult. Moreover, the mechanisms of action of VISTA are still incompletely understood. Its immunosuppressive roles are not always redundant with other checkpoints like PD-1 or CTLA-4. Consequently, designing combination therapies that avoid excessive immune activation while providing synergy remains challenging. From a safety standpoint, early clinical trials of VISTA inhibitors, including CA-170, have shown manageable toxicity profiles, potentially leading to autoimmune or inflammatory adverse events. Lastly, the lack of robust pharmacodynamic markers to assess target engagement and immune modulation in patients has slowed trial progress. Many ongoing trials remain in early phases, and evidence of clinical efficacy is still limited, necessitating more mechanistic studies and refined patient stratification strategies. As result, ongoing trials assess not only safety and pharmacodynamics but also biomarker-driven patient stratification, aiming to identify populations most likely to benefit from VISTA blockade (170–177).

CD40 is a member of the tumor necrosis factor receptor (TNFR) superfamily and is expressed on a variety of antigen-presenting cells (APCs) (178). Its ligand, CD40L (CD154), is primarily expressed on activated CD4⁺ T cells (179). Engagement of CD40 on myeloid cells triggers a cascade of intracellular signaling events involving NF-κB, MAPK, and PI3K/AKT pathways, leading to upregulation of co-stimulatory molecules, cytokine secretion, and enhanced antigen presentation (179–181). In the TME, CD40 signaling reprograms TAMs and dendritic cells from a suppressive, tolerogenic state to an inflammatory, immunostimulatory phenotype. This shift supports the recruitment and activation of effector T cells, especially CD8⁺ cytotoxic T lymphocytes, and promotes tumor cell killing through both direct and indirect mechanisms (182, 183).

CD40 agonists are a class of immunotherapeutic agents designed to mimic CD40L, which activate CD40-expressing myeloid cells within the TME. These agents include agonistic monoclonal antibodies (e.g., selicrelumab (184), APX005M (185), CDX-1140 (186)) and CD40L fusion proteins, which engage CD40 and promote pro-inflammatory remodeling of the TME (187). Preclinical studies have shown that CD40 agonists can eradicate established tumors, even in the absence of checkpoint blockade, by bridging innate and adaptive immunity (188).

In pancreatic ductal adenocarcinoma (PDAC), CD40 agonists have demonstrated the ability to reprogram tumor-associated macrophages (TAMs) into tumoricidal cells that facilitate stromal remodeling and promote T cell–independent tumor regression. In a landmark mouse model study, CD40 agonist therapy alone induced substantial tumor shrinkage without requiring PD-1 or CTLA-4 checkpoint inhibition, highlighting a potent innate immune mechanism (180). In melanoma, CD40 activation enhances dendritic cell (DC) maturation and cross-presentation of tumor antigens, leading to robust CD8⁺ T cell–mediated cytotoxicity. These effects persist even in the absence of additional checkpoint modulation, underscoring CD40’s ability to prime adaptive immunity (181–183). In bladder cancer, preclinical studies using ADC-1013, a human CD40 agonistic antibody, have shown eradication of subcutaneous tumors and development of long-term protective memory responses. These effects were CD8⁺ T cell–dependent but did not require co-administration of checkpoint inhibitors (181–183). In lymphoma, CD40 ligation on malignant B cells leads directly to apoptosis and enhances their immunogenicity, triggering cytotoxic T cell responses capable of eradicating disease. These outcomes occurred independently of PD-1 or CTLA-4 blockade, making CD40 agonists a promising standalone immunotherapy for hematologic malignancies (181). In colorectal cancer, CD40 agonists have been shown to improve antigen presentation by tumor-resident DCs and promote T cell infiltration and expansion within the tumor microenvironment (182, 183). This bridging of innate and adaptive arms led to tumor regression even without additional immune checkpoint therapies. In non–small cell lung cancer (NSCLC) and breast cancer, CD40 stimulation has been associated with enhanced macrophage activation and expansion of effector T cells in murine models, contributing to tumor control (184, 188).

In clinical trials, CD40 agonists have demonstrated tolerable safety profiles, with some showing partial responses and disease stabilization in patients with pancreatic cancer, melanoma, and non-small cell lung cancer (NSCLC) (189–192). In pancreatic ductal adenocarcinoma (PDAC), CD40 agonist therapy—particularly when combined with chemotherapy—has demonstrated disease stabilization and improved immune cell infiltration, as observed in early-phase trials using APX005M and selicrelumab (189). In melanoma, agents such as CP-870,893 have induced partial responses and enhanced T cell activation in patients, with manageable infusion-related adverse events (190, 191). In non–small cell lung cancer (NSCLC), CD40 agonists used as monotherapy or in combination with immune checkpoint inhibitors have led to stable disease in a subset of patients, while enhancing dendritic cell function and T cell priming (190, 191). These findings support the rationale for integrating CD40 agonists into combination regimens to potentiate anti-tumor immunity in immunologically cold tumors.

CD40 agonists have shown promise in enhancing anti-tumor immunity by activating antigen-presenting cells and bridging innate and adaptive immune responses. However, their clinical development has been limited by safety concerns, especially related to cytokine release syndrome (CRS). Systemic CD40 activation can lead to excessive production of pro-inflammatory cytokines (e.g., TNF-α, IL-6), causing flu-like symptoms, hypotension, and liver enzyme elevations, even at low doses (192, 193). Another challenge lies in achieving the right balance of immune activation without triggering off-target inflammation. Because CD40 is broadly expressed on immune and non-immune cells systemic delivery of agonistic antibodies may result in non-specific tissue toxicity, especially in the liver and spleen (180, 194, 195).Furthermore, not all patients respond to CD40 agonists, and responses are often transient. The lack of reliable predictive biomarkers makes it difficult to select appropriate patients or monitor therapeutic response in real time (192). Finally, CD40 agonists may not work well as monotherapies in many solid tumors and often require combination with chemotherapy or checkpoint blockade to achieve meaningful efficacy (180, 192–195).

Given the broad immunostimulatory effects of CD40 agonists, their use in combination therapies is a major area of clinical exploration. One of the most promising strategies is combining CD40 agonists with immune checkpoint inhibitors (195–197). CD40 activation primes and expands T cell responses, while checkpoint blockade sustains T cell activity by preventing exhaustion (195–197).CD40 agonists are also being tested in combination with chemotherapy, which can provide a source of tumor-associated antigens (TAAs) via immunogenic cell death and enhance antigen cross-presentation (198). Other combinations under investigation include CD40 agonists with radiation therapy, TLR agonists, STING activators, or cancer vaccines, all of which can further boost antigen release and immune activation (198–201). Ultimately, the goal is to develop multi-pronged immunotherapeutic regimens where CD40 agonism serves as the immune ignition switch, enabling and enhancing the efficacy of co-administered treatments.