Unlocking new frontiers: novel immune targets for next-generation cancer immunotherapy

Abstract

Cancer immunotherapy represents a transformative strategy in modern oncology, utilizing the body’s immune system to recognize and eliminate malignant cells with precision. Unlike traditional therapies, which often directly target the tumor, immunotherapy enhances the immune system’s inherent ability to differentiate between healthy and cancerous cells. The advent of immune checkpoint inhibitors (ICIs), particularly those targeting the PD-1/PD-L1 and CTLA-4 pathways, has marked a significant breakthrough in this field. However, the therapeutic landscape is still challenged by issues such as the development of resistance mechanisms, heterogeneity in patient responses, and the limited efficacy of current ICIs across all tumor types. Given these challenges, there is a critical need to identify and validate new immune targets that can synergize with existing therapies or function independently to overcome resistance and improve patient outcomes. This review provides a comprehensive overview of the latest research efforts focused on uncovering novel immune targets. By expanding the repertoire of immune targets, these discoveries aim to enhance the effectiveness of cancer immunotherapy, offering hope for more personalized and resilient treatment options. The integration of these novel targets into clinical practice could not only extend the benefits of immunotherapy to a broader spectrum of cancers but also mitigate some of the current limitations, paving the way for more durable and effective therapeutic strategies in the fight against cancer.

INTRODUCTION

Cancer immunotherapy represents a groundbreaking strategy in contemporary oncology, utilizing the body’s immune system to target and eliminate malignant cells. Unlike traditional treatments that directly target tumors, immunotherapy aims to stimulate or enhance the immune system’s ability to recognize and eliminate cancer cells, offering the potential for more durable and targeted therapeutic outcomes [1]. Immunotherapy, including adoptive cell transfer and immune checkpoint inhibitors (ICIs), is a cancer treatment that harnesses the power of the immune system to target and eliminate tumor cells. Used either on its own or alongside traditional treatments such as radiotherapy and chemotherapy, this approach has become a standard and highly successful option for treating many cancers [2]. Notably, ICIs that target PD-1/PD-L1 and CTLA-4 have gained prominence in clinical practice [2]. These therapies have achieved significant success by effectively releasing the brakes on the immune response, allowing the immune system to detect and attack tumors that previously evaded detection. Despite these advancements, challenges remain, such as the emergence of resistance mechanisms and the varying effectiveness of treatment across different types of cancer [1,2]. Consequently, there is a pressing need to identify novel immune targets that can complement existing therapies, broaden the spectrum of cancers that can be effectively treated, and improve overall treatment outcomes.

Emerging immune checkpoints refer to newly identified inhibitory receptors that regulate immune responses, particularly in the tumor microenvironment (TME). These checkpoints, similar to well-established ones like PD-1 and CTLA-4, act as negative regulators of T-cell activation, preventing excessive immune responses but often contributing to immune evasion by tumors [3,4]. Examples include LAG-3 (lymphocyte-activation gene 3), TIM-3 (T-cell immunoglobulin [Ig] and mucin-domain containing-3), and VISTA (V-domain Ig suppressor of T cell activation). These molecules are being explored as potential therapeutic targets to enhance anti-tumor immunity when blocked with checkpoint inhibitors [3,4].

Novel co-stimulatory molecules, in contrast, are immune receptors that promote T-cell activation and enhance anti-tumor responses when engaged [5]. These molecules act as positive regulators of immune function and are critical in the context of cancer immunotherapy. Unlike immune checkpoints that suppress T-cell function, co-stimulatory molecules amplify T-cell proliferation, cytokine production, and cytotoxic activity [6]. Examples include OX40 (CD134), ICOS (inducible T-cell co-stimulator), and 4-1BB (CD137). Agonistic antibodies targeting these receptors are being developed to boost immune responses in cancer treatment [5,6].

This review critically examines the latest advancements in the field, focusing on the discovery and exploration of new immune targets for cancer immunotherapy.

EMERGING IMMUNE CHECKPOINTS

Emerging immune checkpoints represent a new frontier in cancer immunotherapy, offering novel targets to enhance the body’s immune response against tumors. Immune checkpoints like TIGIT (T-cell immunoreceptor with Ig and immunoreceptor tyrosine-based inhibitory motif [ITIM] domains), LAG-3, VISTA, TIM-3, and others, are increasingly being recognized for their significance in regulating immune responses within the TME (Table 1) [3]. These emerging targets are drawing attention due to their potential to influence the effectiveness of cancer immunotherapies by modulating the immune system’s ability to recognize and attack tumor cells (Fig. 1) [3,7]. By targeting these novel checkpoints, either alone or in combination with existing therapies, there is potential to overcome resistance mechanisms, treat a broader range of cancers, and ultimately improve patient outcomes.

Table 1.

Emerging immune checkpoints, ligands, and their immunoregulatory roles

Immune checkpoint	Ligand (s)	Mode of action
LAG-3	MHC class II, galectin-3	Inhibits T-cell activation and proliferation by binding to MHC class II
TIGIT	CD155, CD112, CD113	Suppresses T-cell and NK cell activity through interaction with its ligands
TIM-3	Galectin-9, CEACAM1, HMGB1	Promotes T-cell exhaustion and immune tolerance by interacting with ligands
VISTA	VSIG-3	Negatively regulates T-cell activation and cytokine production
B7-H3	Unknown (possible CD276)	Inhibits T-cell function and may promote tumor immune evasion
BTLA	HVEM	Inhibits T-cell and B-cell activation, contributing to immune tolerance
CD160	HVEM, MHC class I	Inhibits T-cell and NK cell activation through binding to ligands
CD96	CD155	Competes with TIGIT for ligand binding, suppressing NK cell activity
2B4 (CD244)	CD48	Modulates NK and T-cell activity through receptor-ligand interaction
PD-1H (VISTA)	VSIG-3	Inhibits T-cell responses, contributing to immune evasion
HHLA2	TMIGD2, CD28H	Regulates T-cell function, potentially inhibiting immune responses
SIRPα	CD47	Acts as a “don’t eat me” signal, inhibiting phagocytosis by macrophages

Fig. 1.

Emerging immune checkpoints in cancer immunotherapy. Interaction of emerging immune checkpoint receptors and their respective ligands. Various immune checkpoint molecules expressed on T cells are shown with their ligands on APCs or tumor cells. (A) TIGIT and CD226 bind to the same ligands, CD112 and CD155. CD226 is a co-stimulatory receptor whereas TIGIT is a co-inhibitory receptor. TIGIT binds with CD112/CD155 with higher affinity than CD226 leading to the suppression of T cell and NK cell activity, thereby contributing to immune evasion by tumors. (B) LAG-3 binds to MHC-II to inhibit CD4-dependent T cell function with its cytoplasmic domain. TME-derived galectin-3 and LSECtin bind with LAG-3 to inhibit T cell function, which requires the KIEELE motif in the LAG-3 cytoplasmic domain. (C) B7 family receptors (B7-H3, B7-H4, HHLA2) and their known and unknown ligands are inhibitory molecules which mediate various mechanisms to evade tumor-antigen-specific T-cell immunity, including T-cell apoptosis, anergy and exhaustion. (D) TIM-3 is expressed in both T cells and innate immune cells, with four known ligands including carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1), galectin-9, high mobility group box 1 (HMBG1), and phosphatidylserine (PS). In the absence of ligands, BAT3 binds to unphosphorylated Ty256/263 in TIM-3 cytoplasmic domain and recruits active Lck to deliver stimulatory signal in T cells. Interaction with galectin-9/CEACAM1 leads to PS of TIM-3 Ty256/263 and the subsequent abolishment of BAT3 binding. Thus, functioning as an inhibitory receptor and contributing to immune tolerance and anti-tumor immunity suppression. (E) CD47 interacts with SIRPα, acting as a “don’t eat me” signal to prevent macrophages and phagocytes from engulfing cancer cells. Siglec-15 interacts with sialylated ligands, modulating immune responses in the TME and contributing to immune evasion. (F) VISTA serves dual immunosuppressive roles as both a ligand on tumor cells/APCs with PSGL-1 being its receptor on T cells and a receptor on T cells with VSIG3 as its ligand. (G) BTLA interacts with HVEM on APC/tumor cells causing NF-kb activation. ITIM and ITSM in BTLA recruit SHP1/SHP2 to inhibit both TCR and CD28 signaling. APC, antigen-presenting cell; MHC, major histocompatibility complex; TCR, T cell receptor; NK, natural killer; TIGIT, T-cell immunoreceptor with Ig and ITIM domains; ITSM, immunoreceptor tyrosine-based switch motif; LAG-3, lymphocyte-activation gene 3; HHLA2, HERV-H LTR-associating 2; mTOR, mechanistic (or mammalian) target of rapamycin; HIF, hypoxia-inducible factor; IL, interleukin; IFN, interferon; TME, tumor microenvironment; TIM-3, T-cell immunoglobulin and mucin domain containing-3; HMGB, high mobility group box; Siglec-15, sialic acid-binding Ig-like lectin 15; SIRPα, signal regulatory protein alpha; VISTA, V-domain immunoglobulin suppressor of T-cell activation; PKC, protein kinase C; PSGL-1, P-selectin glycoprotein ligand-1; BTLA, B- and T-lymphocyte attenuator; VSIG3, and V-Set and Ig domain containing 3; HVEM, herpesvirus entry mediator; SHP2, Src-homology-2-containing protein tyrosine phosphatases-2; TNF, tumor necrosis factor.

T-cell immunoreceptor with Ig and ITIM domains

TIGIT was first identified in 2009 by researchers at Washington University and was initially named WUCAM (Washington University Cell Adhesion Molecule). It was later referred to as Vstm3 and V-Set and Ig domain containing (VSIG)9, with studies demonstrating its immunosuppressive role in various cancers [3,8]. However, expression of TIGIT is weak on naive T cells [9]. TIGIT is composed of an extracellular Ig variable domain, a type I transmembrane domain, and a short intracellular domain containing one ITIM and one Ig tyrosine tail (ITT)-like motif [9]. TIGIT is expressed on natural killer (NK) cells and T cells, including CD4+ T cells, CD8+ T cells, and regulatory T cells (Tregs) [10]. While TIGIT expression is typically low in naive cells, both T cells and NK cells have been shown to upregulate TIGIT upon activation [11].

Its primary role is to regulate immune responses, maintaining a balance to prevent overactivation that could lead to autoimmunity [11]. TIGIT competes with the co-stimulatory receptor CD226 (DNAM-1) for binding to the same ligands, primarily CD155 and, to a lesser extent, CD112 and CD113, which are expressed on antigen-presenting cells (APCs) including dendritic cells, macrophages [12], and various tumor cells including melanoma, colon cancer, pancreatic cancer, lung adenocarcinoma, and glioblastoma [13,14].

When TIGIT binds to CD155 on APCs or tumor cells, it transmits inhibitory signals to the T cells and NK cells, leading to a suppressed immune response. This suppression helps tumors evade the immune system, contributing to tumor growth and progression [12]. The engagement of TIGIT with CD155 also inhibits the activation and function of CD226 (DNAM-1) in a cell-intrinsic manner, further dampening immune responses [13].

Elevated levels of TIGIT have been observed in the cellular microenvironment of various cancers including non-small cell lung cancer (NSCLC), colorectal adenocarcinoma, gastric cancer, breast cancer, melanoma, multiple myeloma (MM) and acute myeloid leukemia (AML) [12], correlating with an unfavorable prognosis for cancer patients. Numerous studies have documented increased TIGIT expression on CD8+ T cells, alongside reports of elevated TIGIT levels on tumor-infiltrating Tregs and NK cells [15]. Several studies have revealed that high TIGIT expression on tumor-infiltrating lymphocytes (TILs) correlates with poor clinical outcomes in cancer [16,17]. Sun et al. [18] showed that, high TIGIT expression in lung adenocarcinoma was linked to advanced TNM staging, lymphoid metastasis, distant metastasis, and low expression of anti-tumor immunity-related genes. A study by Liu et al. [19] revealed that, in patients with hepatocellular carcinoma, high TIGIT expression in CD8+ T-cell populations in peripheral blood is inversely correlated with survival. Further, in melanoma patients, an elevated TIGIT/CD226 ratio in Tregs is associated with higher Treg frequencies In tumors and poorer clinical outcomes [20]. In endometrial cancer, increased levels of TIGIT on NK cells residing within tumors have been linked to the severity of the disease [21]. A study by Kong et al. [22], noted that, TIGIT expression on CD8+ T cells from peripheral blood collected from patients with AML was increased and was associated with poor prognosis. However, as revealed by Ma [23], increased TIGIT expression in gastric cancer appears to be a positive indicator. It is associated with an active immune landscape, improved survival, greater sensitivity to immunotherapy, and a favorable prognosis. Patients with high TIGIT expression respond better to immunotherapy compared to those with low TIGIT expression. Clinical trials are ongoing to evaluate the safety and efficacy of TIGIT blockade, both as monotherapy and in combination with PD-1/PD-L1 inhibitors, in various types of cancer (Table 2).

Table 2.

Clinical trials evaluating TIGIT blockade in cancer treatment

Clinical Trial ID	Treatment	Cancer type	Key findings	Phase
NCT04952597 AdvanTIG-204	Ociperlimab (anti-TIGIT)+tislelizumab (PD-1 inhibitor)+chemoradiotherapy (cCRT) or tislelizumab+cCRT	Untreated limited-stage SCLC	Tislelizumab+cCRT yielded a trend of improvement in PFS and ORR vs. cCRT; addition of ociperlimab did not show detectable improvement	II
NCT04995523 ARTEMIDE-01	Rilvegostomig (AZD2936), an anti-TIGIT/anti-PD-1 bispecific antibody	Advanced or metastatic NSCLC	Preliminary results: ORR of 29% in PD-L1 TPS 1%–49% and 61.8% in TPS ≥50%, with median PFS of 6.0 and 10.2 months, respectively	I/II
NCT04746924 AdvanTIG-302	Ociperlimab (BGB-A1217) (anti-TIGIT monoclonal antibody)+tislelizumab or pembrolizumab	Unresectable or metastatic NSCLC	On going	III
NCT03563716 CITYSCAPE	Tiragolumab (TIGIT inhibitor)+atezolizumab vs. atezolizumab alone	unresectable or metastatic PD-L1-selected NSCLC	Improved ORR, PFS, and OS in PD-L1-positive NSCLC, tolerable safety profile	II
NCT04256421 SKYSCRAPER-02	Tiragolumab+atezolizumab+chemotherapy (carboplatin and etoposide) or atezolizumab+chemotherapy	Untreated extensive-stage (ES) SCLC	Addition of tiragolumab did not improve OS or PFS in ES-SCLC	III
NCT04294810 SKYSCRAPER-01	Tiragolumab+atezolizumab or atezolizumab	Unresectable, or metastatic NSCLC	No improvement in PFS or OS in PD-L1–high NSCLC but was well tolerated	III
NCT04672356	IBI939 (tamgiblimab), anti-TIGIT monoclonal antibody+sintilimab (PD-1 blocker)	Advanced lung cancer	manageable safety profile and showed promising preliminary efficacy	I
NCT04543617 (SKYSCRAPER-07)	Tiragolumab+atezolizumab vs. placebo	Locally advanced esophageal SCC	On going	III
NCT04047862 (AdvanTIG-105)	Ociperlimab+tislelizumab (BGB-A317)	Locally advanced, or metastatic solid tumors	Well tolerated with an ORR of 10%, DoR of 3.6 months, and DCR of 50%	I

TIGIT, T-cell immunoreceptor with Ig and ITIM domains; SCLC, small cell lung cancer; PFS, progression-free survival; ORR, objective response rate; NSCLC, non-small cell lung cancer; TPS, tumor proportion score; OS, overall survival; SCC, squamous cell carcinoma; DoR, duration of response; DCR, disease control rate.

lymphocyte-activation gene 3

LAG-3 is an approximately 55 kDa type I transmembrane glycoprotein with four Ig-like domains, an interconnecting peptide, and an intracellular inhibitory signaling region, expressed on activated T cells, some B cells, Tregs, NK cells, plasmacytoid dendritic cells, and neurons, and regulated epigenetically [24]. Research on LAG-3 knockout mice and LAG-3 antibodies has shown that LAG-3 primarily plays a role in negatively regulating the activation, proliferation, effector function, and homeostasis of T cells [24,25]. LAG-3 has structural similarities to the CD4 co-receptor, including a similar domain architecture and approximately 25% amino acid sequence identity. It binds to major histocompatibility complex (MHC) class II but has distinct functional properties [24].

The primary ligand for LAG-3 is MHC class II molecules, expressed on APCs such as dendritic cells, macrophages, and B cells [26]. Other ligands include galectin-3, fibrinogen-like protein 1, α-synuclein, and LSECtin [26]. LAG-3 binds MHC class II with higher affinity than CD4, disrupting CD4–MHC class II interactions and transmitting inhibitory signals that suppress T-cell proliferation and cytokine production, maintaining immune homeostasis [24]. In cancer, LAG-3 contributes to immune evasion by suppressing anti-tumor responses, making it a key target for immunotherapy. Co-expression of LAG-3 and PD-1 indicates severe T-cell dysfunction and resistance to anti-PD-1/PD-L1 therapies. Studies show high LAG-3 expression correlates with poor prognosis in triple-negative breast cancer (TNBC), particularly post-neoadjuvant chemotherapy [24,27].

LAG-3 targeting molecules include: anti-LAG-3 monoclonal antibodies, bispecific molecules, LAG-3 fusion protein, and CAR-T cells [24]. Wierz et al. [28] documented that, dual blockade of PD-1 and LAG-3 immune checkpoints restricts tumor development in a murine model of chronic lymphocytic leukemia. In mouse models, Thudium et al. [29], documented that simultaneous blockade of LAG-3 and PD-1 using surrogate antibodies led to enhanced anti-tumor activity that surpassed the effects observed with blockade of either receptor alone. In a study by Matsuzaki et al. [30], dual blockade of LAG-3 and PD-1 during T-cell priming significantly enhanced the proliferation and cytokine production of NY-ESO-1 (“cancer-testis” antigen) -specific CD8+ T cells in epithelial ovarian cancer. Clinical trials are ongoing to evaluate the safety and efficacy of LAG-3 inhibitors, both as monotherapy and in combination (Table 3). In March 2022, the U.S. Food and Drug Administration (FDA) approved the fixed-dose combination of relatlimab and nivolumab for treating unresectable or metastatic melanoma in adult patients and pediatric patients aged 12 years and older, weighing at least 40 kg [31]. Relatlimab, the first LAG-3 inhibitor to be approved, marks the third ICI to enter clinical practice following PD-1 and CTLA-4 inhibitors [31].

Table 3.

Clinical trials of LAG-3 inhibitors

Clinical Trial	Intervention	Cancer type	Key findings	Phase
NCT05064059 (KEYFORM-007)	Favezelimab (MK-4280)	Metastatic colorectal cancer	Discontinued	III
NCT05508867 (KEYFORM-008)	Favezelimab (MK-4280)	Relapsed or refractory classical Hodgkin lymphoma	Discontinued	III
NCT00351949	IMP321 (LAG-3 Ig fusion protein)	Stage IV renal cell carcinoma	Induced CD8+ T cell activation, high doses reduced tumor growth and improved PFS	I
NCT02460224	Ieramilimab (LAG525) alone or in combination with spartalizumab (PDR001)	Advanced/metastatic solid tumors	Well tolerated, modest anti-tumor activity	I/II
NCT03470922 (RELATIVITY-047)	Relatlimab (LAG-3 inhibitor)+nivolumab (PD-1 blocker)	Unresectable or metastatic melanoma	Superior PFS compared to nivolumab monotherapy, leading to FDA approval in March 2022	II/III

LAG-3, lymphocyte-activation gene 3; PFS, progression-free survival; FDA, Food and Drug Administration.

V-domain Ig suppressor of T cell activation

VISTA (gene Vsir) is an emerging immune checkpoint receptor that has garnered attention for its role in regulating immune responses, particularly within the TME [32]. VISTA is predominantly expressed on myeloid cells, including monocytes, macrophages, dendritic cells, microglia, neutrophils, and tumoral cells as well as on certain subsets of TILs [33]. Human VISTA has several confirmed binding partners (ligands) with immunosuppressive functions including P-selectin glycoprotein ligand-1 (PSGL-1),VSIG3, and galectin-9 [34].

The main role of VISTA is to uphold an immunosuppressive environment, which is essential for preventing excessive immune responses, thereby maintaining the body’s homeostasis and protecting against autoimmune tissue damage [32]. However, in the context of cancer, this immunosuppressive function can be hijacked by tumors to evade immune detection and destruction. VISTA achieves this by delivering inhibitory signals to T cells, dampening their activation, proliferation, and effector functions [32]. Studies have demonstrated that tumor-infiltrating immune cells, such as CD11b+Gr1+ myeloid cells and FoxP3+ Tregs, can exhibit increased expression of VISTA, thereby dampening anti-tumoral immune responses. Additionally, the hypoxic conditions within the TME can induce overexpression of VISTA, facilitating immune evasion by tumor cells [35,36].

VISTA is structurally similar to other immune checkpoint molecules but operates through distinct mechanisms [32]. It can act both as a receptor and as a ligand, engaging in interactions that suppress T cell activity [34]. When VISTA is expressed on APCs, it interacts with counter-receptors on T cells, leading to the inhibition of T cell activation. This interaction effectively suppresses the immune response by preventing the T cells from mounting a robust attack against antigens, including those presented by tumor cells [34]. Conversely, when VISTA is expressed on T cells, it can receive inhibitory signals from its ligands present on APCs. This bidirectional inhibitory signaling further enhances immune suppression by dampening the activation and function of T cells [34].

VISTA is often significantly upregulated in tumor-infiltrating immune cells across various cancers, including NSCLC, pancreatic adenocarcinoma, renal cell carcinoma (RCC), colorectal cancers, TNBC, melanoma, oral squamous cell carcinoma and in AML [33,37]. However, the significance of VISTA expression in the TME for patient survival remains controversial, and further research is required to assess VISTA expression and function within the TME. Recent studies on NSCLC, esophageal adenocarcinoma, endometrial carcinoma, and breast cancer have confirmed that high levels of VISTA expression in immune cells are associated with a better prognosis [38]. However, a study by Kuklinski et al. [39] on cutaneous melanoma found a negative correlation between VISTA expression in immune cells and prognosis. Similarly in ovarian cancers, Liao et al. [40], observed that high expression of VISTA on immune cell was significantly associated with poor prognosis. In RCC, the presence of VISTA-positive immune cells in the venous tumor thrombus, but not in the primary RCC, was associated with a poor prognosis [41]. Additionally, recent observations have revealed that VISTA can also be expressed on tumor cells in various cancers, including ovarian, endometrial, gastric cancer, hepatocellular carcinoma, colon cancer and NSCLC [33].

Preclinical studies have demonstrated that blocking VISTA can reinvigorate exhausted T cells and promote their proliferation and cytotoxic activity against tumor cells. Blocking VISTA by anti-VISTA mAb can also inhibit the recruitment of myeloid-derived suppressor cells (MDSCs) and increases dendritic cells [42]. In a study by Le Mercier et al. [36], VISTA mAb treatment enhanced the infiltration, proliferation, and effector function of tumor-reactive T cells within the TME. VISTA blockade also altered the suppressive nature of the TME. Additionally, VISTA blockade impaired the suppressive function and reduced the emergence of tumor-specific Foxp3+CD4+ Tregs. In the CT26 colorectal cancer model, anti-VISTA monotherapy reduced small tumor growth, while its addition to anti-PD-1/CTLA-4 overcame resistance in large tumors, leading to rejection in half the cases. Single-cell RNA sequencing revealed that anti-VISTA activated CD8+ T-cell pathways by enhancing co-stimulatory gene expression and suppressing quiescence regulators [35]. This has led to increased interest in developing VISTA-targeted therapies as a novel approach in cancer immunotherapy. The first anti-VISTA antibody to undergo human testing is CI-8993. CI-8993 is designed with putative binding sites at four residues within the C-C′ loop of VISTA and has been demonstrated to block interactions with both PSGL-1 and VSIG3. It features an active IgG1 Fc domain that enhances interactions with Fc gamma receptors and myeloid cells, supporting antibody-dependent cell cytotoxicity (ADCC) [42]. Further anti-VISTA antibodies BMS767) and HMBD-002 are in preclinical development [43]. Mehta et al. [44], used yeast surface display to engineer an anti-VISTA antibody, SG7, which binds with high affinity to VISTA in mice, humans, and cynomolgus monkeys. SG7, as a monotherapy and even more effectively in combination with anti-PD1, slows tumor growth in multiple syngeneic mouse models.

In particular, combining VISTA inhibitors with other immunotherapies, such as PD-1/PD-L1/CTLA-4 inhibitors, holds significant promise [34]. In a murine colon cancer model, Dr. Lines and her team showed that anti-VISTA therapy increased immune cell infiltration in the TME, including NK, CD45+, CD8+, and CD4+ T cells. Combining anti-VISTA with anti-PD-1 and anti-CTLA-4 further enhanced tumor suppression and reduced myeloid cell-mediated immunosuppression [35]. The rationale for combination therapy is that simultaneously targeting multiple immune checkpoints can produce synergistic effects, leading to a more robust and sustained anti-tumor immune response [45]. For instance, while PD-1/PD-L1 inhibitors work by lifting the “brakes” on T cells, VISTA inhibitors can further enhance T cell activation by disrupting additional inhibitory pathways. Clinical trials are currently underway to evaluate the safety and efficacy of VISTA inhibitors, both as monotherapies and in combination with other checkpoint inhibitors [46]. Several VISTA-targeting inhibitors are being tested in phase I and II trials in patients with advanced, metastatic or unresectable solid tumors (NCT04475523) (NCT05082610) (NCT04564417). A phase 1 dose-escalation study (NCT04475523) is currently being conducted in patients with advanced, treatment-resistant solid tumors to evaluate CI-8993 (anti-VISTA antibody). So far, the safety data suggests that the treatment is manageable, with no dose-limiting side effects noted up to a dose of 0.6 mg/kg [47]. In a phase II TRIAL, oral dual inhibitor targeting both VISTA and PD-L1, known as CA-170, exhibited a clinical benefit rate of 75% and achieved a median progression-free survival (PFS) of 19.5 weeks in a cohort of eight previously treated non-squamous NSCLC patients [48].

T-cell Ig and mucin-domain containing-3

TIM-3 is a TIM family immunoregulatory protein with an Ig V domain, mucin stalk, transmembrane domain, and cytoplasmic tail, regulated by NFAT signaling in CD8+ T cells and transcription factors NFIL3, T-bet, and STAT3 [49]. TIM-3 is an inhibitory receptor expressed on various interferon (IFN)-γ producing immune cells, including CD4+ T cells, CD8+ T cells, NK cells, FoxP3+ Treg, dendritic cells, macrophages and monocytes. TIM-3 plays a critical role in regulating immune responses and maintaining immune homeostasis. It is involved in suppressing immune responses and inducing immune tolerance, primarily by depleting CD8+ T cells. While this function can help prevent autoimmunity, it is detrimental in the context of cancer [49]. TIM-3 protein also has a role in efferocytosis [50]. Fourcade et al. [20] observed that in melanoma patients, the upregulation of TIM-3, in conjunction with PD-1, results in a subset of CD8+ T cells that are highly non-responsive.

TIM-3 interacts with several ligands, the most well-studied of which is galectin-9. Other ligands include phosphatidylserine (PtdSer), high mobility group protein B1 (HMGB1), and cancer-embryonic antigen cell adhesion molecule 1 (CEACAM1). These ligands attach to different regions on the TIM-3 extracellular Ig V domain [49]. The interaction between TIM-3 and its ligands transmits inhibitory signals to immune cells, leading to the suppression of their activation and effector functions [51]. For instance, the binding of galectin-9 to TIM-3 on T cells can induce cell death or exhaustion, a state where T cells lose their ability to proliferate and produce cytokines in response to antigen stimulation [52].

In cancer, TIM-3 expression is often upregulated on T cells within the TME. This upregulation contributes to the immune evasion mechanisms of tumors by suppressing the anti-tumor immune response. High levels of TIM-3 expression are associated with the suppression of T cell responses and T cell dysfunction, which is a gradual loss of T cell function in a hierarchical manner during tumor development [53]. TIM-3 activates the interleukin (IL)-6-STAT3 pathway, which directly suppresses CD4+ T cells and inhibits Th1 polarization [53]. TIM-3 is also expressed on other immune cells in the TME, including Tregs and MDSCs, further contributing to an immunosuppressive environment [54].

Blocking TIM-3 has emerged as a promising strategy to rejuvenate exhausted T cells and enhance anti-tumor immunity [53]. By inhibiting the interaction between TIM-3 and its ligands, TIM-3 blockade can restore the function of exhausted T cells, increasing their proliferation, cytokine production, and cytotoxic activity. Preclinical studies have shown that TIM-3 blockade can enhance anti-tumor immunity. A study by Ngiow et al. [55] observed that anti-mouse TIM-3 monoclonal antibodies (mAb) used against experimental and carcinogen-induced tumors enhance CD8+ and CD4+ T cells IFN-γ-mediated anti-tumor immunity and suppress established tumors. A study by Kikushige and Miyamoto, in xenograft models reconstituted with human AML leukemic stem cells or hematopoietic stem cells, a TIM-3 mouse IgG2a antibody with cytotoxic activities eliminated AML leukemic stem cells in vivo, while preserving normal human hematopoiesis [56].

When used in combination with other ICIs, such as PD-1/PD-L1 inhibitors, TIM-3 blockade has demonstrated synergistic effects [57]. The rationale for this combination therapy is that targeting multiple inhibitory pathways can produce a more comprehensive and potent reactivation of T cells. PD-1/PD-L1 inhibitors lift the suppression mediated by the PD-1 pathway, while TIM-3 blockade further enhances T cell function by targeting a different inhibitory mechanism [58]. Sakuishi et al. [57] found that Tim-3+PD-1+ T cells dominate TILs in mice with solid tumors and exhibit severe exhaustion with impaired cytokine production and proliferation. Dual blockade of Tim-3 and PD-1 more effectively reversed T cell exhaustion and restored anti-tumor immunity than targeting either pathway alone [57]. Zhou et al. [59] identified a distinct phenotype of exhausted T cells in mice with advanced AML, characterized by concurrent expression of Tim-3 and PD-1. This co-expression escalated as AML advanced. PD-1+ Tim-3+ CD8+ T cells exhibited impaired production of IFN-γ, tumor necrosis factor (TNF)-α, and IL-2 in response to AML cells expressing PD-L1 and Tim-3 ligand (galectin-9). In a work by Fourcade et al. [60], dual blockade of PD-1 and Tim-3 enhanced the expansion and cytokine production of vaccine-induced CD8+ T cells in vitro. In a murine model of ovarian cancer, Guo et al. [61] noted that either anti-TIM-3 or CD137 mAb alone was unable to prevent tumor progression in mice bearing established tumor, however, combined anti-TIM-3/CD137 mAb significantly inhibited the growth of these tumors with 60% of mice tumor free 90 days after tumor inoculation. Therapeutic efficacy was associated with a systemic immune response with memory and antigen specificity. Ongoing clinical trials are assessing the safety and efficacy of TIM-3 inhibitors, both as standalone treatments, and in combination with other checkpoint inhibitors (Table 4). The results of these trials will provide important insights into the potential of TIM-3 inhibitors as a new class of immunotherapies.

Table 4.

Ongoing clinical trials TIM-3 inhibitors

Clinical Trial ID	Intervention	Cancer type	Status	Phase
NCT04823624	MBG453 (sabatolimab), an anti-TIM-3 mAb	Lower-risk myelodysplastic syndromes	Ongoing	II
NCT04623892	TQB2618, an anti-TIM-3 mAb	Solid tumors	Ongoing	I
NCT03489343	Sym023, an anti-TIM-3 mAb	Advanced solid tumor malignancies or lymphomas	Ongoing	I
NCT03680508	TSR-022 (cobolimab)+TSR-042 (dostarlimab)	Advanced hepatocellular carcinoma	Ongoing	II
NCT04139902	Cobolimab (TSR-022), a TIM-3 binding antibody, and dostarlimab (TSR-042), a PD-1 binding antibody	Resectable stage III or oligometastatic stage IV melanoma	Ongoing	II
NCT03708328	Lomvastomig (RO7121661), a bispecific antibody targeting both PD-1 and TIM-3	Advanced and/or metastatic solid tumors	Ongoing	I

TIM-3, T-cell immunoglobulin and mucin-domain containing-3.

B- and T-lymphocyte attenuator

B- and T-lymphocyte attenuator (BTLA; CD272) is an inhibitory receptor that belongs to the CD28 Ig superfamily (IgSF). It is structurally similar to PD-1 and CTLA-4, with an IgC-like extracellular domain, transmembrane domain, and cytoplasmic region containing ITIM, ITSM, and a Grb2-binding motif [62]. It is expressed on various immune cells, including T cells, B cells, dendritic cells, and NK cells [62]. The primary function of BTLA is to maintain immune homeostasis and prevent overactivation of the immune system, which can lead to autoimmune diseases [63].

BTLA interacts with its ligand, HVEM. The N-terminal cysteine-rich domain CRD1 of HVEM binds to single IgC domain of BTLA on the cell surface in a 1-to-1 ratio [64]. BTLA-HVEM interaction leads to inhibition of both CD28 and CD3ζ phosphorylation resulting in the inhibition of T-cell activation [65]. Hence, the binding of BTLA to HVEM delivers inhibitory signals to immune cells. UL144 viral protein was identified as the second BTLA ligand, although it has a 5-fold lower affinity to BTLA; however, it activates similar inhibitory signaling pathways [66]. Although UL144 has a lower binding affinity for BTLA compared to HVEM, it more effectively restricts T-cell proliferation [67]. Interestingly, recent research also indicates that BTLA expressed on APCs can function as a co-stimulatory ligand for HVEM present on CD8+ T cells [67]. HVEM is broadly expressed on many cell types, including T cells, B cells, and APCs [64].

In the context of cancer, the inhibitory signals mediated by BTLA can contribute to the immune evasion mechanisms of tumors [62]. BTLA is expressed in TILs and is often associated with a diminished anti-tumor immune response [62]. In a study by Oguro et al. [68], it was found that a higher density ratio of BTLA+ cells to CD8+ T cells in TME serves as an independent indicator of poor prognosis in gallbladder cancer patients. Additionally, the upregulation of BTLA in cancer tissues is implicated in the suppression of anti-tumor immunity. T cells from lung cancer patients also exhibit elevated BTLA expression [69]. Studies have also found increased BTLA expression in T cells from melanoma patients [70]. Chen et al. [71] observed that BTLA expression in cancerous tissues can serve as a predictor of poor outcomes in patients with epithelial ovarian cancer. Sekar et al. [72] noted that type I NK T cells highly express BTLA in murine autochthonous mammary tumors. In diffuse large B-cell lymphoma, BTLA+ T cells show increased expression of other checkpoint molecules (PD-1, TIM-3, LAG-3), reduced cytolytic activity, a poorly differentiated phenotype, and enhanced proliferative capacity. Elevated BTLA levels correlate with advanced disease stages [73]. However, some studies have yielded contradictory findings. For example, research by Song and Wu [74] indicated that BTLA levels were lower in colorectal cancer tissues compared to matched non-carcinoma tissues. Interestingly, BTLA appears to be capable of both inhibitory and survival signaling, suggesting it may have context-specific roles in TILs [63].

BTLA blockade enhances T cell activation by disrupting BTLA-HVEM signaling, boosting proliferation, cytokine production, and cytotoxicity, ultimately improving tumor control and regression in preclinical models [71]. A study on murine autochthonous mammary tumors found that BTLA-neutralizing antibodies inhibit tumor growth and reduce pulmonary metastasis [72]. In a preclinical study by Lasaro et al. [75], it was observed that blocking the BTLA/CD160 pathway along led to the regression of large, established tumor masses in a genetically engineered murine thyroid adenocarcinoma model. Additionally, blocking BTLA is being investigated in combination with other immunotherapies, particularly ICIs like PD-1/PD-L1 inhibitors. A study by Sun et al. [76] found that, dual inhibition of BTLA and PD-1 enhances the therapeutic efficacy of paclitaxel on intraperitoneally disseminated tumors. Chen et al. [71] observed that BTLA blockade enhances cancer therapy by inhibiting IL-6/IL-10-induced CD19high B lymphocytes, both in animal models and in vitro studies.

In 2019, the FDA approved icatolimab (TAB004/JS004), the world’s first-in-class anti-BTLA humanized IgG4 monoclonal antibody, for clinical trials [62]. Currently, BTLA inhibitors are undergoing clinical trials to evaluate the safety and efficacy, both as monotherapies and in combination with other checkpoint inhibitors (Table 5).

Table 5.

Clinical trials evaluating BTLA inhibitors

Clinical Trial ID	Intervention	Cancer type	Key findings/status	Phase
NCT04773951	Tifcemalimab (JS004), recombinant mAb targeting BTLA+toripalimab (anti-PD-1)	Advanced solid tumors	Preliminary findings: favorable safety profile and preliminary clinical efficacy, one partial response and six cases of stable disease	I
NCT05000684	Tifcemalimab+toripalimab (anti-PD-1)	Advanced lung cancer	Preliminary findings: well-tolerated, preliminary efficacy data indicate potential clinical activity	I/II
NCT04929080	Tifcemalimab+ JS001 (anti-PD-1)	Advanced head and neck cancers	Ongoing	I/II
NCT04137900	Icatolimab (also known as tifcemalimab or TAB004)	Advanced solid tumors	Well tolerated in all doses evaluated and showed preliminary clinical efficacy as a monotherapy	I
NCT05891080	Tifcemalimab+toripalimab+with platinum-based doublet chemotherapy	Stage III NSCLC	Ongoing	II
NCT05789069	HFB200603 (anti-BTLA mAb)+tislelizumab (anti-PD-1)	Advanced solid tumors	Ongoing	I

BTLA, B- and T-lymphocyte attenuator; NSCLC, non-small cell lung carcinoma.

B7-H3 (CD276) and B7-H4

B7-H3 (CD276) and B7-H4 (B7S1, B7x, Vtcn1) are immune checkpoint molecules belonging to the B7 family, playing essential roles in immune regulation by providing co-stimulatory or co-inhibitory signals to T cells and other immune cells to maintain immune homeostasis [77].

B7-H3

B7-H3, a type I transmembrane protein, is widely expressed in tumor cells and immune cells within the TME but is rarely found in normal tissues [77]. Its expression varies across cancer types but is significantly upregulated in several solid tumors, including colorectal, gastric, esophageal, lung, pancreatic, prostate, ovarian, and breast cancers [78], making it a promising target for immunotherapy. Initially at the time of its discovery, it was reported that B7-H3 exerted a co-stimulating effect on the proliferation of both CD4+ and CD8+ T cells [79], but latter on a larger majority of studies has revealed that B7-H3 induces a more robust immune evasive effect when deregulated in cancers. Suh et al. discovered that murine B7-H3 inhibits T cell proliferation [80]. Veenstra et al. [81] in their study observed that B7-H3 is responsible for providing a negative co-stimulatory signal. A study conducted by Cong et al. [82] found that elevated levels of CD24 and B7-H3 were associated with a poor prognosis in breast cancer patients. Another study identified B7-H1 and B7-H3 as independent predictors of poor prognosis in patients with NSCLC [83]. In cancer, B7-H3 can exert immunosuppressive effects through multiple mechanisms. It can deliver inhibitory signals to T cells, leading to decreased T cell activation, proliferation, and cytokine production [77]. Moreover, B7-H3 expression on tumor cells can directly promote their survival and resistance to immune-mediated destruction [84]. B7-H3 is also seen to induce drug resistance in various cancers [85]. Targeting B7-H3 with specific inhibitors or blocking antibodies has shown promise in preclinical studies and early clinical trials. In vitro and in vivo studies have shown, that experimental depletion or blocking of B7-H3, enhance the anti-tumor immune response and inhibit tumor cell proliferation and migration [86,87]. Attempts are being made to target B7-H3 via., monoclonal antibodies, bispecific antibodies, antibody-drug conjugates, CAR-T cells and CAR NK cells, and B7-H3 small-molecule inhibitors [84,87]. Through these modalities, several clinical trials are in progress as discussed in Table 6. Preliminary data from some of the clinical trials have shown promising results [88,89].

Table 6.

Clinical trials targeting B7-H3

Clinical Trial ID	Intervention	Cancer type	Key findings/status	Phase
NCT01391143	Enoblituzumab (MGA271), an Fc-optimized mAb B7-H3	Refractory B7-H3-expressing cancers	Well-tolerated with no dose-limiting toxicities. Preliminary evidence of antitumor activity was observed	I
NCT02923180	Enoblituzumab	Localized intermediate-and high-risk prostate cancer	Generally well-tolerated, 66% of patients achieved undetectable PSA levels at 12 months	II
NCT02982941	Enoblituzumab	Pediatric, B7-H3-expressing relapsed or refractory solid tumors	Well-tolerated in pediatric patients. Preliminary antitumor activity seen, details awaited	I
NCT02381314	Enoblituzumab+ipilimumab	Refractory B7-H3-expressing cancers	Preliminary antitumor activity seen, details awaited. Generally well-tolerated	I
NCT02628535	MGD009 (obrindatamab), a d B7-H3 x CD3 dual-affinity re-targeting (DART) protein	Unresectable or metastatic B7-H3-expressing neoplasms	FDA imposed a partial hold (2018) on the study due to hepatic adverse events. Trial was terminated in November 2019 due to administrative reasons	I
NCT03406949	MGD009 (orlotamab), B7-H3 × CD3 DART protein	Relapsed or refractory B7-H3-expressing tumors	Ongoing	I
NCT05280470	Ifinatamab deruxtecan (I-DXd; DS-7300), ADC targeting B7-H3	Extensive-stage SCLC	Ongoing	III
NCT04145622 (IDeate-PanTumor01)	ifinatamab deruxtecan (I-DXd; DS-7300)	Advanced solid malignant tumors	Ongoing	I/II
NCT03729596	MGC018, an anti-B7-H3 ADC,+MGA012 (anti-PD-1 antibody)	Advanced solid tumors	In the mCRPC cohort, 54% of patients experienced PSA reductions of 50% or greater. Partial responses were observed in both mCRPC and NSCLC patients. Safety: manageable, with neutropenia being the only Grade 3 or higher treatment-related adverse	I/II
NCT02475213	Enoblituzumab (MGA271), an anti-B7-H3 mAb+pembrolizumab (anti-PD-1 therapy)	Advanced B7-H3-expressing cancers	Acceptable safety profile. ORR were observed in 33.3% of checkpoint inhibitor-naive SCCHN patients and 35.7% of checkpoint inhibitor-naive NSCLC patients	I
NCT04385173	B7-H3 CAR-T therapy	Recurrent and refractory glioblastoma	Ongoing	I/II
NCT04077866	B7-H3 CAR-T cell therapy+temozolomide cycles/temozolomide	Recurrent or Refractory Glioblastoma	Ongoing	I/II
NCT04185038 (BrainChild-03)	B7-H3-specific CAR-T cell	Diffuse intrinsic pontine glioma/diffuse midline glioma and recurrent or refractory pediatric CNS tumors	Ongoing	I
NCT04432649 (GIMI-IRB-20009)	4SCAR-276 CAR-T cell therapy	CD276 (B7-H3) positive solid tumors	Ongoing	I/II
NCT04637503	4SCAR-T cell therapy targeting three tumor-associated antigens: GD2, PSMA, and CD276 (B7-H3)	Relapsed or refractory neuroblastoma (NB)	Ongoing	I/II
NCT05143151	CD276 (B7-H3)-targeted CAR-T cell therapy	Unresectable, locally advanced recurrent, or metastatic pancreatic cancer	Ongoing	I/II
NCT05190185	TAA06, CAR-T cell targeting B7-H3 (CD276)	Advanced solid tumors	Ongoing	I
NCT04630769	FT516, a NK cell therapy+IL-2+enoblituzumab	Recurrent ovarian, fallopian tube, or primary peritoneal cancer	Ongoing	I
NCT04022213	Omburtamab, a radioimmunotherapy agent	DSRCT and other B7-H3-positive solid tumors in the peritoneum	Ongoing	II
NCT04167618	Omburtamab, a radioimmunotherapy agent	Recurrent or refractory medulloblastoma	Ongoing	I/II
NCT04743661	cRIT with 131I-omburtamab (targeted radiation to tumor cells expressing B7-H3 (CD276))+chemotherapy regimen of irinotecan, temozolomide, and bevacizumab	Pediatric patients with recurrent medulloblastoma	Ongoing	II
NCT03275402	Intraventricular Crit with 131-I-omburtamabto	Pediatric patients with CNS or leptomeningeal metastases from neuroblastoma	Ongoing	II/III

PSA, prostate-specific antigen; FDA, Food and Drug Administration; ADC, antibody-drug conjugate; SCLC, small cell lung cancer; mCRPC, metastatic castration-resistant prostate cancer; NSCLC, non-small cell lung cancer; ORR, objective response rate; SCCHN, squamous cell carcinoma of the head and neck; CAR-T, chimeric antigen receptor T-cell; NK, natural killer; DSRCT, desmoplastic small round cell tumors; cRIT, conjugated radioimmunotherapy; CNS, central nervous system.

B7-H4

B7-H4 is another member of the B7 family that functions as a negative regulator of T cell responses [90]. It is primarily expressed on tumor cells and certain immune cells, such as macrophages and dendritic cells, within the TME [90]. Similar to B7-H3, B7-H4 expression is upregulated in several cancers, including melanoma, colorectal cancer, prostate cancer, ovarian cancer, breast cancer, urothelial cancer, lung cancer, and RCC [91]. Its expression is associated with various adverse clinicopathological features, such as larger tumor size, higher primary tumor classification, elevated TNM clinical stage, reduced tumor-infiltrating lymphocyte counts, and decreased survival rates [92], B7-H4 inhibits T cell activation and effector functions by disrupting ERK, JNK, and AKT signaling, leading to reduced proliferation and IL-2 production [93]. Additionally, B7-H4 can promote tumor immune evasion by modulating the function of APCs and enhancing the recruitment of Tregs [94]. A study by Sica et al. [95], noted that administration of B7-H4 Ig into mice impairs antigen-specific T cell responses whereas blockade of endogenous B7-H4 by specific monoclonal antibody promotes T cell responses. In their study, Zhou et al. [90] observed that B7-H4 was highly expressed in breast carcinomas. They found that B7-H4 surface expression on tumor cells was inversely correlated with CD8+ T-lymphocyte infiltration, and these tumor cells exhibited enhanced growth in immunocompetent mice. In human cervical cancer, a study found that B7-H4 promotes the proliferation of Tregs and the secretion of IL-10 and transforming growth factor (TGF)-β1 [96]. Similarly, in colorectal cancer, Treg proliferation was increased along with cancer tolerance under influence of B7-H4 [97]. Various approaches, including mAb and fusion proteins that block the B7-H4 pathway, are being developed and evaluated in preclinical and clinical settings [98].

CD47

CD47, or integrin-associated protein, is an IgSF member that plays a key role in immune evasion, particularly in cancer. It consists of an N-terminal IgV domain, five transmembrane domains, and a short cytoplasmic tail [99]. CD47 serves as a “don’t eat me” signal on red blood cells, preventing their phagocytosis until its loss marks them for clearance by splenic macrophages [100]. It also protects platelets and lymphocytes from rapid elimination. Binding to signal regulatory protein alpha (SIRPα) triggers phosphorylation of inhibitory motifs, activating SHP-1 and SHP-2 phosphatases, which suppress phagocytosis by preventing myosin-IIA accumulation at the phagocytic synapse [100]. Additionally, CD47 interacts with integrins and thrombospondin-1 and is expressed on innate immune cells like macrophages, dendritic cells, and monocytes [101].

CD47 is universally expressed in both normal and malignant tissues [99]. CD47 was initially recognized as a tumor antigen in human ovarian cancer during the 1980s [102]. Since then, increased expression of CD47 has been observed in various cancers, including colorectal cancer, gastric cancer, bladder cancer, and lung squamous carcinoma, among others [103]. Upregulation of CD47 helps tumors evade immune surveillance and clearance [99]. Further, it is involved in numerous cellular functions, playing a crucial role in processes such as proliferation, apoptosis, adhesion, and migration [104].

In recent years, targeting the CD47-SIRPα pathway has emerged as a promising strategy in cancer immunotherapy. Preclinical studies have shown that blocking CD47 using mAb or possibly with a recombinant SIRPα protein that can bind and block CD47 can reverse this “don’t eat me” signal, enabling macrophages to recognize and engulf cancer cells effectively [99,101]. Anti-CD47 antibodies can facilitate the elimination of tumor cells through several mechanisms, including phagocytes, Fc-dependent mechanisms such as ADCC and complement-dependent cytotoxicity, as well as direct induction of apoptosis [99,101]. Studies on mouse models have shown that administering a blocking anti-human CD47 antibody, resulted in the elimination of AML and acute lymphoblastic leukemia cells in both the peripheral blood and bone marrow. This treatment led to long-term remissions in some cases [105]. Chan et al. [106] noted that, blocking CD47 with a mAb resulted in macrophage engulfment of bladder cancer cells in vitro. A study by Chao et al. [107] reported that the anti-CD47 antibody synergizes with rituximab to promote phagocytosis and eradicate non-Hodgkin lymphoma (NHL) cells, in human NHL-engrafted mice. Antagonistic antibodies against SIRPα significantly enhance the in vitro killing of trastuzumab-opsonized Her2/Neu-positive breast cancer cells by phagocytes [108]. Combination approaches that target the CD47-SIRPα pathway hold significant therapeutic promise. This includes incorporating antibodies against CD47-SIRPα into combination therapies with other therapeutic antibodies, chemo-radiation therapy, agents that enhance macrophage activity, adjuvant therapy to prevent metastasis or standard cancer treatments [101]. One of the most advanced CD47 inhibitors in clinical development is magrolimab, a humanized 5F9 antibody (also known as Hu5F9-G4). Hu5F9-G4 induced robust macrophage-mediated phagocytosis of primary human AML cells in vitro and achieved complete eradication of human AML in vivo, resulting in long-term disease-free survival of patient-derived xenografts [109]. Clinical trials investigating magrolimab (Hu5F9-G4) have demonstrated encouraging results across various hematologic malignancies, including AML, myelodysplastic syndrome, NHL, and MM (NCT02641002) (NCT02367196) [110,111]. TTI-622, a bispecific antibody targeting both CD47 and CD19, is currently undergoing phase I clinical trials for patients with relapsed or refractory B-cell lymphoma and chronic lymphocytic leukemia [112]. Moreover, CD47 blockade is also being evaluated in solid tumors, where it has shown potential in enhancing anti-tumor immunity and improving responses to other therapies, such as chemotherapy and ICIs. ALX148, a fusion protein targeting CD47, is currently under evaluation in multiple clinical trials, including a phase I/II trial for patients with advanced solid tumors [99]. KSI-3716, a monoclonal antibody targeting CD47, is currently undergoing phase I/II trials for patients with advanced solid tumors [113].

Sialic acid-binding Ig-like lectin 15

Sialic acid-binding Ig-like lectin 15 (Siglec-15) is an emerging immune checkpoint receptor that has gained attention for its role in regulating immune responses within the TME. It contains only a V-set Ig structural domain and a C2-set Ig, which has a high structural similarity to PD-L1 [114].

Siglec-15 is predominantly expressed on tumor-associated macrophages (TAMs) and other myeloid cells within the TME. It is also expressed on cancer cells [114]. TAMs are a crucial component of the innate immune system and play diverse roles in tumor progression, including promoting immunosuppression and facilitating tumor growth [115]. The expression of Siglec-15 on TAMs contributes to the creation of an immunosuppressive TME by dampening anti-tumor immune responses [114]. Chen et al. [116] found that high Siglec-15 expression, primarily on peritumoral CD68+ macrophages, correlated with poor survival in glioma patients and GL261 tumor models. Activated macrophages expressing Siglec-15 have been shown to enhance TGF-β secretion via the DAP12-Syk pathway and suppress CD4+ and CD8+ T cell activity by binding to their respective receptors. This process contributes to tumor progression by modulating intratumoral microenvironments through TGF-β [117]. Another study found that inhibiting Siglec-15 expression in cultured osteosarcoma cells reduced the DUSP1-mediated suppression of p38/MAPK and JNK/MAPK expression. Additionally, DUSP1 overexpression facilitated the proliferation, migration, and invasion of osteosarcoma cells. The group concluded that Siglec-15 promotes the malignant progression of osteosarcoma cells by suppressing DUSP1-mediated suppression of the MAPK pathway [118]. Data analyzed from 13 observational studies involving 1,376 patients revealed that Siglec-15 expression is significantly associated with poor outcomes in human solid tumors [119]. Bioinformatics analyses revealed that elevated Siglec-15 levels are associated with poor clinical prognosis and shorter recurrence times in glioma patients [114]. However, some studies have observed an opposite or equivocal relationship regarding prognosis. According to Chen et al. [120], positivity for Siglec-15 is associated with a good prognosis in pancreatic ductal adenocarcinoma.

The ligands of Siglec-15 are less characterized. The interaction of Siglec-15 with its ligands (CD44, CD11b, Muc5B) or other components of the T cell receptor (TCR) and immune synapse, triggers inhibitory signals that suppress immune activation and effector functions [121]. This includes reducing the production of pro-inflammatory cytokines and inhibiting the cytotoxic activity of T cells and NK cells against tumor cells. Moreover, Siglec-15 expression on TAMs can promote their polarization towards an M2-like phenotype, which further supports tumor growth and immune evasion [122].

In the context of cancer, targeting Siglec-15 has emerged as a potential therapeutic strategy to reprogram the immunosuppressive TME and enhance anti-tumor immunity [123]. Siglec15 is emerging as a promising immunotherapeutic target in glial, bladder, breast, gastric, colon, and pancreatic cancers [124,125]. Preclinical studies have shown that blocking Siglec-15 using mAb or other inhibitors can alleviate immune suppression and promote immune surveillance against cancer cells. A study by Sun et al. [123] documented that Siglec-15 blocking mAbs significantly slowed down the tumor growth in mice. According to a study by Wang et al. [114], genetic ablation or antibody blockade of Siglec-15 enhances anti-tumor immunity within the TME and inhibits tumor growth in certain mouse models. By inhibiting Siglec-15, researchers also aim to shift TAMs towards an M1-like phenotype, which is associated with anti-tumor activity and the promotion of T cell-mediated immune responses [114]. Clinical trials are underway to evaluate the safety, efficacy, and optimal dosing of Siglec-15 inhibitors as monotherapy and in combination with other immunotherapies or standard treatments (Table 7).

Table 7.

Clinical trials targeting Siglec-15

Clinical Trial ID	Intervention	Cancer type	Key findings/status	Phase
NCT03665285	NC318, a mAb targeting Siglec-15	Advanced or metastatic solid tumors	Well-tolerated, disease control rate of 38% was reported, including one CR, three PR, and 28 cases of stable disease	I/II
NCT04699123	NC318 with pembrolizumab	Advanced NSCLC	Ongoing	II
NCT05718557	PYX-106, a mAb targeting Siglec-15	Advanced solid tumors	Ongoing	I

Siglec-15, sialic acid-binding immunoglobulin-like lectin 15; CR, complete response; PR, partial response; NSCLC, non-small cell lung cancer.

CD96 (T cell activation, increased late expression)

CD96, also known as T cell activation increased late expression (TACTILE), is an emerging immune checkpoint receptor that plays a significant role in regulating immune responses, particularly in the context of cancer immunotherapy [126]. It is a type I transmembrane glycoprotein expressed on various immune cells, including T cells, NK cells, and subsets of dendritic cells and monocytes [126]. The primary function of CD96 is to modulate immune cell activation and effector functions through its interactions with ligands such as CD155 (Necl5, poliovirus receptor) [127]. CD155 is expressed on both tumor cells and APCs, where it acts as a binding partner for CD96, as well as for other immune checkpoint receptors like TIGIT and CD226 (DNAM-1) [128]. CD96 shares similarities with TIGIT in terms of its binding competition with CD226 for CD155 [128]. This competition is crucial in regulating the balance between stimulatory and inhibitory signals that control immune responses. CD226 is an activated receptor on the surface of T and NK cells. In vivo experiments have demonstrated that CD226 mediates the phosphorylation of FOXO1 and activates NK cells through its interaction with CD155-expressing tumor cells [129]. CD112 is usually downregulated in tumor tissue [130]. However, when CD96 binds to CD155, it delivers inhibitory signals that dampen immune cell activation and cytotoxicity, thereby contributing to immune evasion by tumors [131].

In cancer, elevated CD96 expression has been observed on exhausted T cells and dysfunctional NK cells within the TME, correlating with reduced anti-tumor immunity and poorer prognosis [126]. A study by Xu et al. [132] demonstrated that, high infiltration of CD96+ cells predicted poor prognosis and reduced survival benefits from fluorouracil-based adjuvant chemotherapy in the Zhongshan Hospital (ZSHS) cohort. A study by Sun et al. [133] documented that human CD96 is associated with NK cell exhaustion and can predict the prognosis of human hepatocellular carcinoma. The Cancer Genome Atlas data revealed that CD96 expression was notably elevated in high-grade gliomas, isocitrate dehydrogenase wild-type gliomas, and gliomas of the mesenchymal molecular subtype [134]. However, few studies demonstrate that CD96 functions as a co-stimulatory receptor to enhance CD8+ T cell activation and effector responses [135].

Therefore, targeting CD96 presents an attractive strategy to restore immune cell function and enhance anti-tumor responses. Preclinical studies have demonstrated that inhibiting CD96 can lead to enhanced NK cell and T cell activity against tumor cells [126]. Combining CD96 inhibition with other immunotherapies, such as PD-1/PD-L1 inhibitors or TIGIT blockade, holds promise for synergistically enhancing anti-tumor immunity. In three different tumor models, a study demonstrated that co-blockade of CD96 and PD-1 effectively inhibited lung metastases, significantly enhancing local NK cell IFN-γ production and infiltration [136]. A compelling study by Mittal et al. [137] demonstrated that combining anti-CD96 with anti-PD1 and anti-TIGIT therapies yielded superior anti-tumor responses in various experimental mouse tumor models. These results were observed regardless of the Fc receptor engagement ability of the anti-TIGIT isotype. Clinical trials are underway to evaluate the safety, efficacy, and therapeutic potential of CD96 inhibitors as monotherapy and in combination with other treatments (NCT04446351) (NCT03739710) [138].

CD112R (poliovirus receptor-related Ig domain containing)

CD112R, also known as PVRIG (poliovirus receptor-related Ig domain-protein), is an emerging immune checkpoint receptor that plays a significant role in regulating immune responses, particularly in the context of cancer immunotherapy. It is a type I transmembrane glycoprotein expressed on various immune cells, including T cells, NK cells, and subsets of myeloid cells [139]. The primary ligand for CD112R is CD112 (PVRL2, nectin-2), a member of the nectin family of adhesion molecules. CD112 is expressed on both tumor cells and APCs, where it serves as a binding partner for CD112R. The interaction between CD112R and CD112 delivers inhibitory signals that modulate immune cell activation and effector functions [139].

In cancer, CD112R expression has been observed on exhausted T cells and dysfunctional NK cells within the TME. Studies have shown that CD112R exhibits high expression on NK cells in ovarian, endometrial, kidney, prostate, lung, and breast cancers [140]. In a group of 60 ovarian cancer patients, elevated CD112 expression correlated with lymph node metastasis and residual tumor presence following surgery [140]. High expression of CD112R has also been noted in liver metastases from colorectal cancer [141]. Karabulut et al. [142] noted that serum levels of CD112R have diagnostic value, with higher levels correlating with an adverse prognostic impact on PFS in patients with early-stage colorectal cancer. A study by Murter et al. [143] showed that enhanced CD8+ T-cell effector function inhibited tumor growth more effectively in PVRIG−/− mice compared to wild-type mice. Preclinical studies have demonstrated that CD112R blockade independently or in combination with other therapies, can lead to enhanced cytotoxicity and cytokine production by T cells and NK cells, which are crucial for recognizing and eliminating cancer cells. Blocking CD112R enhanced T-cell function, in an ex vivo study using tumor-derived T cells. When combined with TIGIT or PD-1 blockade, this effect was further enhanced [144]. A preclinical study by Xue et al. [145] noted that, IBI352g4a, a novel humanized anti-PVRIG antibody with Fc-competent function, induced significant NK cell activation in TILs (single dose), and also T-cell activation was observed after the second dose by blocking the interaction between PVRIG and its ligand PVRL2. Blockade of CD112R separately, or in combination with TIGIT signaling sensitizes human NK cell functions in post-trastuzumab therapy resistant breast cancer [146]. Clinical trials are currently underway to evaluate the safety, efficacy, and therapeutic potential of CD112R inhibitors (Table 8).

Table 8.

Clinical trials to evaluate CD112R inhibitors

Clinical Trial ID	Treatment	Cancer type	Status	Phase
NCT03667716	COM701 (anti-CD112R mAb)	Advanced solid tumors	Ongoing	I
NCT04570839	COM701+nivolumab (anti-PD-1 mAb)	Advanced solid tumors	Ongoing	I/II
NCT04491967	COM701+nivolumab+BMS-986207 (anti-TIGIT mAb)	Advanced solid tumors	Ongoing	I/II

HERV-H LTR-associating 2

HERV-H LTR-associating 2 (HHLA2) is a member of the B7 family of immune checkpoint molecules that has garnered attention for its role in regulating immune responses, particularly in the context of cancer immunotherapy [147]. Studies have shown that HHLA2 plays a role in immune regulation by interacting with its receptors on T cells and other immune cells. It is constitutively expressed on the surface of human monocytes and can be induced on B cells. HHLA2 molecule is also highly expressed in TAMs [147].

HHLA2 interacts with KIR3DL3 (killer cell Ig-like receptor with three Ig domains and a long cytoplasmic tail) and TMIGD2 (T cell membrane protein with Ig and ITIM domains 2) to exhibit inhibitory and stimulatory functions, respectively. Despite the presence of TMIDG2, the inhibitory function mediated by the KIR3DL3-HHLA2 interaction predominates [148]. Therefore, tumors may escape immune surveillance through the KIR3DL3-HHLA2 pathway by suppressing CD4 and CD8 T-cell activation and effector functions in the presence of T-cell receptor signaling. This includes reducing T-cell proliferation, cytokine production, and cytotoxic activity against tumor cells [149]. KIR3DL3-HHLA2 pathway also inhibits the cytotoxicity of NK cell [148].

HHLA2 expression has been observed on various types of tumors, including RCC, pancreatic cancer, melanoma, hepatocellular carcinoma, ovarian cancer, gastric cancer, colorectal cancer, thyroid cancer, breast cancer, and lung cancer [150,151]. Elevated levels of HHLA2 have been associated with poorer prognosis, highlighting its role as a potential target for therapeutic intervention [150,152]. However, better survival and prognosis is also reported with overexpression of HHLA2 in some studies [153,154].

Targeting HHLA2 with specific inhibitors or blocking antibodies represents a novel strategy to enhance anti-tumor immunity. Considering the beneficial role of the TMIGD2 receptor, an ideal therapeutic approach would involve selectively blocking the interaction between HHLA2 and KIR3DL3 [149,155]. Monoclonal antibody targeting the HHLA2/KIR3DL3 pathway, which block the inhibitory activity of KIR3DL3 while preserving the immune-stimulatory effects of HHLA2 via TMIGD2 have shown promising results in preclinical studies [155]. A study by Wei et al. [149] revealed that KIR3DL3 blockade inhibits tumor growth in multiple humanized mouse models. However, study by Wang et al. [156] showed targeting TMIGD2 signaling with anti-TMIGD2 mAb diminishes leukemia stem cell self-renewal and decreases leukemia burden in AML patient-derived xenograft models, while having minimal impact on normal hematopoietic stem and progenitor cells. Clinical trials are underway to evaluate the safety, efficacy, and therapeutic potential of HHLA2 inhibitors in various cancers. In July 2023, a multicenter first-in-human study, (phase I clinical trial, NCT06240728) commenced to evaluate NPX887, an antagonistic IgG1 monoclonal antibody targeting HHLA2 (B7-H7)/KIR3DL3 interaction. This antibody aims to reactivate exhausted T and NK cells in HHLA2-positive solid tumors. The trial focuses on recurrent or metastatic solid tumors, including RCC, NSCLC and small cell lung cancer, colorectal cancer, and TNBC.

NOVEL CO-STIMULATORY MOLECULES

Co-stimulatory molecules are a diverse group of proteins expressed on the surface of APCs and other cells involved in immune responses. They play a crucial role in amplifying or reducing (secondary signals), the initial activation signals delivered to T cells by the TCR following its interaction with an antigen/MHC, thereby influencing T cell differentiation and outcome [5]. Hence, co-stimulatory molecules provide secondary signals to T cells in addition to the primary signal delivered through the TCR interaction with antigen-presenting molecules (e.g., MHC molecules) [5,157].

The primary function of co-stimulatory molecules is to ensure that T cell activation occurs appropriately in response to pathogens or other stimuli (Fig. 2). Without adequate co-stimulation, T cells may become tolerant or undergo apoptosis (cell death) instead of becoming activated and mounting an immune response [5]. Traditionally, the best-known co-stimulatory molecules belong to the CD28 family, such as CD80 (B7-1) and CD86 (B7-2), which interact with CD28 on T cells to promote activation [157].

Fig. 2.

Novel co-stimulatory molecules. This diagram illustrates the interaction of various novel co-stimulatory molecules on T cells with their corresponding ligands on antigen-presenting cells (APCs), highlighting their roles in T cell activation and immune response modulation. GITR, glucocorticoid-induced tumor necrosis factor receptor-related protein; ICOS, inducible T cell co-stimulator; DR3, death receptor 3; TCR, T cell receptor; MHC, major histocompatibility complex; IL, interleukin; IFN, interferon; TNF, tumor necrosis factor.

However, ongoing research has identified several novel co-stimulatory molecules that expand our understanding of immune regulation and offer new opportunities for therapeutic interventions (Fig. 2). These novel co-stimulatory molecules can be classified into different families based on their structural and functional characteristics. For example, members of the TNF receptor superfamily (TNFRSF) (e.g., OX40, 4-1BB) and the TNF ligand superfamily (e.g., CD40, CD27) have emerged as important regulators of T cell responses [5,157]. Other families include adhesion molecules (e.g., ICOS, LFA-1) and receptors involved in cytokine signaling (e.g., IL-2R, IL-7R) [157]. These co-stimulatory molecules are mainly categorized into two groups: the “IgSF” include CD28, ICOS, and CD226, and the “TNFRSF” including OX40 receptor (CD134; TNFRSF4), CD27, 4-1BB (CD137; TNFRSF9), and glucocorticoid-induced TNF receptor (GITR)-related protein (CD357; TNFRSF18) [158]. Additionally, cell adhesion molecules including CD2 and LFA-1 (lymphocyte function-associated antigen 1) act as co-stimulatory molecules [159].

The roles of these co-stimulatory molecules vary widely. Some enhance T cell activation and effector functions, promoting immune responses against infections and tumors. Others regulate immune tolerance and prevent excessive immune activation, thereby maintaining immune homeostasis and preventing autoimmune diseases [5].

Inducible T-cell co-stimulator

ICOS (CD278) is a co-stimulatory receptor of the CD28 family expressed on activated T cells and constitutively on FOXP3+CD25+CD4+ Tregs, playing a crucial role in immune regulation [160]. Upon activation, ICOS interacts with its ligand (ICOSL) on APCs and some tumor cells, modulating T cell activation, effector functions, and Treg-mediated suppression. This dual role of the ICOS/ICOSL axis can influence both anti-tumor immunity and immune suppression, mediated through cytokine production such as IL-4, IL-10, and IFN-γ [161].

In the context of cancer immunotherapy, ICOS/ICOSL axis has been demonstrated to promote anti-tumor T cell responses when activated in Th1 and other Teff cells, or to promote protumor responses when triggered in Tregs [160]. Combining ICOS agonists with ICIs, such as antibodies targeting PD-1 or CTLA-4, have shown to potentiate the effect of inhibitory checkpoint blockade in preclinical studies [162]. A study by Fan et al. [162] observed that in mouse models of melanoma and prostate cancer, simultaneous CTLA-4 blockade and ICOS engagement through tumor cell vaccines engineered to express the ICOS ligand enhanced anti-tumor immune responses both quantitatively and qualitatively, significantly improving tumor rejection. Both agonistic (GSK3359609, JTX-2011) and antagonistic (MEDI-570, KY1044) mAbs targeting the ICOS/ICOSL pathway are being explored for cancer immunotherapy in clinical trials [160]. INDUCE-2 (NCT03693612) a phase I/II, open-label clinical trial, in patients with advanced solid tumors, anti-ICOS agonist (feladilimab), administered alone or combined with an anti-CTLA-4 antibody tremelimumab, showed promising outcomes in tolerability, toxicity profile, but showed limited efficacy [163]. The phase I/II ICONIC trial investigated the ICOS agonist vopratelimab, both as a monotherapy and in combination with nivolumab, in patients with advanced solid tumors. The trial demonstrated a favorable safety profile for vopratelimab alone and in combination with nivolumab, particularly in patients with high ICOS CD4 T-cell populations [164]. ICOS mAbs are unlikely to be used as monotherapy because they do not independently induce satisfactory cytotoxic immune responses [160].

OX40 (CD134)

OX40 (also known as CD134 or TNFRSF4) is a co-stimulatory receptor expressed primarily on activated T cells (regulatory T phenotypes constitutively and by effector T cells after activation), belonging to the TNFRSF. Its interaction with its ligand, OX40 ligand (OX40L), which is expressed on APCs and other cell types (vascular endothelial cells, mast cells, and some T cells.), plays a critical role in regulating immune responses [165].

Upon binding to OX40L, OX40 signaling delivers potent co-stimulatory signals to T cells. These signals promote T cell activation, expansion, survival, and differentiation into effector and memory T cells. OX40 signaling also enhances the production of cytokines such as IL-2, IFN-γ, and TNF-α, which are crucial for orchestrating effective immune responses against pathogens and tumors [166].

Agonistic antibodies or other agents (OX40L-Fc fusion proteins, transfected DCs with OX40L mRNA, and tumor cells engineered to express OX40L on their surface, immune-activating recombinant modified vaccinia virus Ankara (rMVA, MVAΔE5R-Flt3L-OX40L), that activate OX40 have shown encouraging result [165,167]. These agents aim to amplify T cell responses within the TME, where immune responses are often suppressed. By enhancing T cell activation and function, OX40 agonists can potentially overcome immune evasion mechanisms employed by tumors and improve the efficacy of anti-cancer immune responses. Fully human IgG1 agonist mAb developed include INCAGN01949, IBI101, GSK3174998 and BMS-986178 [167,168]. Fully human IgG2 agonist Ab developed include ivuxolimab (PF-04518600) and utomilumab (PF-05082566) [169]. mRNA-2752 is a lipid nanoparticle encapsulating mRNAs encoding human OX40L, IL-36γ and IL-23 [170]. MEDI6383 is a human OX40L IgG4P Fc fusion protein. SL-279252 is a dual-sided Fc fusion protein PD1-Fc-OX40L [165]. In a study by Campos Carrascosa et al. [171], treatment with an Fc-engineered αOX40 antibody (αOX40_v12), which has selectively enhanced FcγRIIB affinity, stimulated the expansion of CD4+ and CD8+ TILs in vitro, as well as the secretion of cytokines and chemokines [171]. A study by Reuter et al. [172] demonstrated that, OX40L transgenic Ewing sarcoma cells showed enhanced immune stimulation against Ewing sarcoma cells in combination with IL-2 and stimulation of CD137 [172]. Monotherapy and combination therapies involving OX40 agonists with other immunotherapies, such as ICIs (e.g., anti-PD-1, anti-CTLA-4 antibodies), are actively being explored by clinical trials (Table 9).

Table 9.

Clinical trials targeting OX40

Clinical Trial ID	Intervention against OX40	Cancer type	Status	Phase
NCT02274155	MEDI-6469, an anti-OX40 mAb	Locally advanced HNSCC	MEDI6469 boosted T-cell proliferation, TIL activation, and tumor-reactive CD8+ TILs, with 25% of patients remaining disease-free	Ib
NCT02219724	MOXR0916 (vonlerolizumab), an agonist antibody targeting OX40	Locally advanced or metastatic solid tumors	Manageable safety profile, preliminary evidence of antitumor activity was observed	I
NCT01644968	9B12, mouse mAb	Advanced cancers	12 out of 30 patients experienced regression in at least one metastatic lesion	I
NCT03894618	SL-279252, a bifunctional fusion protein combining PD-1 and OX40L	Advanced solid tumors or lymphomas	1 patient with ocular melanoma achieved a durable partial response, indicating potential antitumor activity	I
NCT01303705	MEDI-6469+cyclophosphamide (+radiation therapy	Metastatic prostate cancer	Awaited	Ib
NCT03390296	PF-04518600 (OX40 agonist mAb)+azacitidine/avelumab/glasdegib/gemtuzumab/ozogamicin	Relapsed or refractory AML	Combination of azacitidine, venetoclax, and gemtuzumab ozogamicin (Arm B) showed the most promise. Patients not previously treated with venetoclax achieved a composite complete remission (CR/CRi) rate of 50%, while those with prior venetoclax exposure had a rate of 22%	Ib/II multi-arm
NCT02315066	Ivuxolimab (PF-04518600), an agonistic Mab+ utomilumab (PF-05082566), a 4-1BB agonist	Locally advanced or metastatic cancers	Antitumor activity was observed in selected patient groups, particularly in those with melanoma resistant to ICI	I
NCT02410512	MOXR0916, an OX40 agonist mAb+atezolizumab	Locally advanced or metastatic solid tumors	Generally well-tolerated. Preliminary antitumor activity was observed. However, the overall response rate was limited	Ib
NCT03336606	MEDI0562, mAb prior to surgical resection	HNSCC or melanoma	Ongoing active	Ib
NCT05105971	BAT6026, mAb	Advanced solid tumors	BAT6026 showed stronger anti-tumor activity compared to PF-04518600 and exhibited a synergistic effect when combined with PD-1 antibodies	I
NCT04714983	DNX-2440, an oncolytic adenovirus	Resectable colorectal liver metastases	Suspended	I
NCT05229601	HFB301001, mAb	Advanced solid tumors	Well-tolerated, Anti-tumor activity have been observed	I
NCT04730843	ES102, mAb	Advanced solid tumors	Preliminary signs of anti-tumor activity were observed, supporting further investigation	I
NCT04648202	FS120, dual-agonist tetravalent bispecific antibody+CD137 (4-1BB)	Advanced malignancy	Ongoing active	I/1b
NCT05263180	EMB-09, bispecific antibody targeting PD-L1 and OX40	Advanced or metastatic solid tumors	Consistent pharmacodynamic response and early efficacy signals	I
NCT01862900	MEDI6469 (anti-OX40)+SBRT	Stage IV breast cancer	Result awaited	I
NCT03831295	BMS 986178 (OX40 agonist antibody)+TLR9 agonist SD-101	Advanced or metastatic solid malignancies	Result awaited	I
NCT03217747	PF-04518600 (an OX40 agonist)+avelumab, an anti-PD-L1 mAb+utomilumab (a 4-1BB agonist), and radiation therapy	Advanced malignancies	Active	I/II
NCT04198766	INBRX-106, a hexavalent OX40 agonist+pembrolizumab	Advanced or metastatic solid tumors	Well tolerated, monotherapy and in combination showed potential antitumor activity	I/II
NCT04991506	ES102, an OX40 agonist,+JS001 (toripalimab), an anti-PD-1	Advanced solid tumors	Active	I/II
NCT04215978	BGB-A445, OX40 agonist mAb+tislelizumab, an anti-PD-1	Advanced solid tumors	Well tolerated, preliminary antitumor activity in both treatment arms	I
NCT05109650	BAT6026, anti-OX40 mAb+BAT1308 (an anti-PD-1 antibody)	Advanced solid tumors	Terminated due to high costs	I

HNSCC, head and neck squamous cell carcinoma; TILs, tumor-infiltrating lymphocytes; AML, acute myeloid leukemia; ICI, immune checkpoint inhibitors; SBRT, stereotactic body radiation therapy.

CD137 (4-1BB)

CD137 (4-1BB, TNFRSF9) is a co-stimulatory receptor in the TNFRSF, primarily expressed on activated T cells (CD4+, CD8+) and NK cells, playing a key role in immune regulation [173]. Upon binding its ligand (4-1BBL) on APCs, it enhances T cell proliferation, survival, and memory formation, with a preferential effect on CD8+ T cells, making it a promising target for cancer immunotherapy [173]. Additionally, CD137 signaling activates dendritic cells and NK cells, amplifies cytokine production (IL-2, IFN-γ, TNF-α), and synergizes with CD28 to promote robust immune responses [174].

CD137 agonists aim to enhance anti-tumor immune responses by stimulating T cell and NK cell cytotoxicity against tumor cells as seen in a study by Gauttier et al. [175]. Further, in a mouse model of malignant melanoma, the anti-CD137 antibody prevents cancer recurrence and metastasis following the removal of primary tumors by expanding antigen-specific memory T lymphocytes [176]. First-generation agonistic antibodies, urelumab (BMS-663513) and utomilumab (PF-05082566) which can activate CD137, had been developed and investigated in preclinical and clinical studies [177]. While it is evident that these anti-4-1BB antibodies are outstanding at curbing tumor growth in diverse in vivo mouse models, a significant hurdle remains: developing a strategy to translate this anti-4-1BB immunotherapy into clinical settings with a manageable toxicity profile [178].

The phase I clinical trial assessing the safety of urelumab (an agonist antibody targeting CD137) revealed significant transaminitis, which was strongly linked to doses of 1 mg/kg or higher [179]. In another phase Ib study, utomilumab (PF-05082566) (also an agonist antibody targeting CD137) in combination with pembrolizumab (MK-3475), only 26.1% of patients had confirmed complete or partial responses, although there was a very low incidence of toxicities [180]. Current research is centered on adjusting the functions of 4-1BB antibodies. Yu et al. [181] created a lower-affinity variant of utomilumab, converting it from an inert antibody into a highly effective agonistic antibody. Between 2017 and 2022, at least 41 4–1BB agonistic drugs have progressed to phase 1 clinical trials, according to published reports and data from the U.S. National Library of Medicine’s clinicaltrials.gov registry, the Chinese Clinical Trial Registry, and the European Union Trial Register, at least 41 4–1BB agonistic drugs have entered phase 1 clinical trials [177].

Glucocorticoid-induced TNF receptor

GITR (TNFRSF18, CD357) is a co-stimulatory receptor expressed on activated T cells and Tregs, with its expression upregulated upon T cell activation [182]. In Tregs, GITR serves as a marker of activation and is linked to suppressive cytokines like TGF-β and IL-10 [182]. GITR interacts with its ligand (GITRL) on APCs and other immune cells, delivering co-stimulatory signals that enhance T cell activation, proliferation, and cytokine production (IL-2, IFN-γ, TNF-α). Notably, GITR activation in Tregs can modulate their suppressive function, potentially enhancing anti-tumor immunity [183]. According to Ronchetti et al. [182], “GITR activation impacts Treg/effector cell interplay in four distinct ways: (1) temporary inhibition of Treg regulatory activity; (2) reduced sensitivity of effector T cells to Treg suppression; (3) killing of Tregs (particularly within solid tumors); and (4) enhanced proliferation and expansion of the Treg compartment.”

In the context of cancer immunotherapy, GITR has emerged as a promising target. Agonistic antibodies or other agents that activate GITR have been developed and are under investigation in preclinical and clinical studies. These GITR agonists aim to boost anti-tumor immune responses by overcoming immune suppression mediated by Tregs within the TME [184]. Also, by stimulating GITR on activated T cells and modulating Tregs, GITR agonists can enhance T cell proliferation, cytokine secretion, and cytotoxic activity against cancer cells [184]. A study by Amoozgar et al. [185] concluded that, although immune checkpoint blockers have been unsuccessful in all phase III glioblastoma (GBM) trials due to Treg activities, targeting GITR in Treg cells with an agonistic antibody (αGITR) promotes CD4 Treg cell differentiation into CD4 effector T cells, reduces Treg cell-mediated suppression of the anti-tumor immune response, and induces potent anti-tumor effector cells in GBM. A study by Schoenhals et al. [186] revealed that GITR therapy overcomes radiation-induced Treg immunosuppression and leads to enhanced effects of radiotherapy, in two tumor 344SQR murine models. Clinical trials are evaluating GITR agonists as monotherapy and in combination with other immunotherapies, such as ICIs (e.g., anti-PD-1, anti-CTLA-4 antibodies). AMG 228 (NCT02437916), BMS-986156 (NCT02598960), MEDI1873 (NCT02583165), and GWN323 (NCT02740270) are various GITR mAbs currently in clinical trials [187]. The first-in-human phase 1 trial (NCT01239134 ) of GITR agonism using the anti-GITR antibody TRX518 demonstrated that it is safe and produces significant immune effects in patients with incurable cancer. The trial team further indicated that there is mechanistic preclinical evidence supporting the rational combination of GITR agonism with checkpoint blockade [188].

CD40 (TNFRSF member 5)

CD40, also referred to as TNFRSF member 5 (TNFRSF5), is an essential co-stimulatory receptor predominantly found on APCs like dendritic cells, macrophages, and B cells. It is also expressed on non-immune cells such as endothelial, epithelial, and mesenchymal cells (including fibroblasts, myofibroblasts, synoviocytes, stellate cells, etc.), as well as on platelets and tumor cells [189]. CD40 was initially identified as a surface marker on bladder carcinoma cells and B cells [190]. When CD40 interacts with its ligand, CD40 ligand (CD40L or CD154), which is primarily expressed on activated T cells, a cascade of signaling events is initiated. This interaction leads to the activation and maturation of APCs, enhancing their ability to present antigens and provide co-stimulatory signals to T cells [191]. CD40 engagement also promotes the secretion of pro-inflammatory cytokines such as IL-12, IL-6, and TNF-α from APCs, which are crucial for promoting T cell activation and differentiation into effector cells [192].

Agonistic antibodies that specifically activate CD40 have been developed and investigated in preclinical and clinical studies [193]. Preclinical studies have demonstrated that CD40 agonists can induce potent anti-tumor immune responses, including increased infiltration of cytotoxic T cells into tumors, enhanced tumor cell killing, and tumor regression. Furthermore, CD40 activation can lead to the generation of long-lasting memory T cells, providing durable protection against tumor recurrence [193,194]. Several phase 1 clinical trials have evaluated CD40 agonists like recombinant CD40L [193,194], CP-870,893 (fully human IgG2 mAb) [194], SGN-40 (humanized IgG1 mAb) [195], initial findings from these studies show objective clinical responses and immune modulation in the absence of significant toxicity [194]. In a phase I clinical trial of the humanized anti-CD40 monoclonal antibody dacetuzumab in refractory or recurrent NHL, monoclonal antibody was well tolerated with encouraging preliminary response data [196].

CD27 (TNFRSF member 7)

CD27, also known as TNFRSF member 7 (TNFRSF7), is a co-stimulatory receptor expressed on various immune cells, normally expressed on CD4+ and CD8+ T cells, NK cells and thymocytes, and on memory B cells (primed B cells) [197]. CD27 is expressed on naive CD4+ and CD8+ T cells, while most other co-stimulatory TNFRs are produced only after T cell activation [198]. Its ligand, CD70 (CD27-L, TNFSF7), is primarily expressed on activated NK cells, APCs such as dendritic cells, and on some subsets of activated T (conventional and regulatory T cells) and B cells. The interaction between CD27 and CD70 plays a critical role in regulating immune responses, particularly in promoting T cell activation and memory formation [199].

When CD27 on T cells engages with CD70, it initiates signaling pathways that enhance T cell activation, survival, and effector function. This includes promoting T cell proliferation and cytokine production, such as IL-2, IFN-γ, and TNF-α [197]. These cytokines are crucial for orchestrating effective immune responses against infections and tumors. In addition to its role in T cell activation, CD27 signaling also contributes to the generation and maintenance of memory T cells. Memory T cells are essential for providing long-lasting immune protection against previously encountered pathogens and tumors [200].

Preclinical research has shown that CD27 agonists can enhance tumor-specific T cell responses, promote tumor regression, and improve survival in animal models. Agonistic anti-CD27 antibodies in a murine model of melanoma, led to reduction in growth of lung metastases and subcutaneous tumors, due to enhanced effector function and persistence, and reduced PD-1 expression of tumor-infiltrating CD8+ T cells [201]. In a study by Sakanishi and Yagita [202] on mice with syngeneic T-cell lymphoma, a non-depleting agonistic mAb against CD27 demonstrated promise for cancer therapy by co-stimulating the induction of tumor-specific cytotoxic T lymphocytes. A study by French et al. [203] discovered that administering agonistic anti-CD27 mAbs without a DC maturation signal completely protected tumor-bearing mice, offering a highly potent method for enhancing anti-tumor T-cell immunity. Yang et al. [204] conducted a study showing that TanCAR-T cells, which target CD70 and B7-H3, displayed improved anti-tumor activity and addressed the issues of antigenic heterogeneity and variability across multiple tumor tissue samples.

The mAb targeting CD27, known as varlilumab (also referred to as CDX-1127 or 1F5), has progressed into clinical trials following promising results in preclinical studies [205]. In transgenic mice expressing hCD27, the fully human IgG1 monoclonal antibody 1F5, which has agonist activity, effectively induced proliferation and cytokine production from hCD27-Tg-derived T cells when combined with TCR stimulation [206]. The combination of CD27 agonists with checkpoint inhibitors, for example, aims to enhance the efficacy of immune checkpoint blockade by augmenting T cell activation and overcoming immunosuppressive mechanisms within tumors. Preliminary data from these trials show promising results (well tolerated with promising biological and early clinical activity) [207].

TNFRSF25 (death receptor 3)

TNFRSF25, or death receptor 3 (DR-3; also known as TRAMP, LARD and WSL-1), is a member of the TNFRSF expressed on various immune cells, including T cells and NK cells [208]. Its ligand, TNF-like ligand 1A (TL1A or TNFSF15), is primarily expressed on APCs and endothelial cells. The interaction between TNFRSF25 and TL1A plays a critical role in regulating immune responses, particularly in modulating T cell and NK cell functions [208]. When TNFRSF25 engages with TL1A, it triggers intracellular signaling pathways that enhance immune cell activation and effector functions. This includes promoting T cell proliferation, survival, and cytokine production, such as IL-2, IFN-γ, and TNF-α. These cytokines are essential for mounting effective immune responses against infections and tumors [209].

In the context of cancer immunotherapy, TNFRSF25 has emerged as a potential therapeutic target [210]. Agonistic antibodies or other agents that activate TNFRSF25 are being investigated in preclinical studies [211]. These TNFRSF25 agonists aim to enhance anti-tumor immune responses by boosting the cytotoxic activity of T cells and NK cells against cancer cells [211]. A study by Slebioda et al. [212] demonstrated that TNFRSF25 agonists (soluble TL1A) in mouse plasmacytomas lead to the elimination of tumor cells in a CD8+ T-cell-dependent manner and render mice immune to subsequent tumor cell challenges. They proposed that TNFRSF25 agonists like soluble TL1A could potentially be used to enhance the immunogenicity of vaccines designed to elicit human anti-tumor CD8+ T cells.

TARGETING THE TME

Targeting the TME has become a central focus in the search for new immune targets in cancer immunotherapy. The TME, which includes various non-cancerous cells, signaling molecules, and the extracellular matrix surrounding the tumor, plays a crucial role in helping tumors evade the immune system [213]. This approach not only weakens the tumor’s defenses but also enhances the effectiveness of existing therapies. As we better understand the complex interactions within the TME, these new targets (Fig. 3) offer hope for more effective and durable cancer treatments, potentially leading to improved patient outcomes.

Fig. 3.

Targeting the tumor microenvironment. This diagram illustrates key pathways and targets within the tumor microenvironment. (A) The adenosine pathway includes CD39 and CD73, which convert ATP to adenosine, leading to immunosuppression. (B) TAM receptors (Tyro3, AXL, Mer) are shown, which mediate immune evasion and tumor progression. (C, D) Neoantigens and T cell receptor (TCR) engineering are highlighted, representing the identification of tumor-specific antigens and the modification of T cells to enhance anti-tumor immune responses.

CD73 and adenosine pathway

The adenosine pathway, involving the enzymes CD73 and CD39, plays a crucial role in shaping the immunosuppressive TME. These enzymes are highly expressed in various cell types within the TME, including tumor cells, endothelial cells, and infiltrating immune cells such as Tregs and stromal cells [214]. Notably, CD73 and CD39 are upregulated in response to adenosine signaling and the hypoxic conditions commonly found in tumors. The generation of adenosine through the CD39/CD73 pathway is a key mechanism underlying the immunosuppressive function of Tregs [214, 215].

The extracellular degradation of ATP (adenosine triphosphate) by CD39 and CD73 contributes significantly to immunosuppression. This process reduces ATP-dependent immune activation and results in the production of adenosine [214,215]. CD73 activity on the cell surface is the rate-limiting step in the production of extracellular adenosine, a process that hinders anti-tumor immunity and supports tumor progression [216]. Adenosine, a nucleoside molecule, exerts its potent immunosuppressive effects by binding to specific receptors on immune cells, primarily the A2A and A2B adenosine receptors. This receptor engagement triggers a cascade of intracellular signaling events that inhibit immune cell activation and effector functions, thus enabling tumors to evade immune detection and destruction [214, 215].

CD73, also known as ecto-5′-nucleotidase, is an is a glycosyl-phosphatidylinositol-linked cell membrane-bound ectoenzyme, encoded by the gene NT5E [217]. Its primary function involves catalyzing the conversion of extracellular AMP (adenosine monophosphate) to adenosine. This enzymatic activity leads to the accumulation of adenosine in the tumor milieu, particularly heightened under conditions of tissue hypoxia and inflammation typical of solid tumors [217]. Adenosine, upon binding to A2A and A2B receptors (A2AR and A2BR) expressed on T cells and NKT cells, monocytes, macrophages, dendritic cells and NK cells, exerts potent immunosuppressive effects. These effects include the suppression of cytotoxic T cell responses, inhibition of dendritic cell maturation and antigen presentation, promotion of Treg cell differentiation and function, and reduction in pro-inflammatory cytokine production such as IFN-γ and TNF-α [218]. A2AR is upregulated in macrophages in response to NF-κB, STAT1 and PPARγ as well as adenosine signaling, and A2AR activation inhibits the secretion of neutrophil chemokines, thereby reducing the inflammatory response [214]. Thus, CD73-mediated adenosine production plays a critical role in fostering an immunosuppressive environment within tumors, contributing to immune evasion and supporting tumor progression. CD73 has been shown to play a role in various cancer processes, such as metastasis, tumor invasion, and increased cell proliferation [219,220].

CD39, also known as ectonucleoside triphosphate diphosphohydrolase-1 or NTPDase 1, is an ectoenzyme prominently expressed on immune cells, including Tregs and subsets of activated T cells [221]. Its primary function involves the hydrolysis of ATP and ADP (adenosine diphosphate) into AMP. This enzymatic activity serves as a critical step in the production of adenosine within the TME. By generating AMP, CD39 acts upstream of CD73, facilitating the subsequent conversion of AMP into adenosine [222].

A significant proportion of cancer patients fail to respond to immunotherapies such as PD-1/PD-L1 and CTLA-4 blockade, indicating that other immunosuppressive pathways may contribute to immune evasion in these non-responding tumors [223]. The adenosinergic pathway, presents a promising new therapeutic approach in cancer immunotherapy, though still in its early stages [221]. Preclinical studies and clinical trial data have shown that targeting this pathway is a viable therapeutic strategy for the future. Small-molecule inhibitors and monoclonal antibodies targeting CD39, CD73 and A2AR have been developed for cancer therapy [214,224]. As small molecules could cross physiologic barriers in TME, they are better than mAb which are macromolecules. Bastid et al. [225] in their study showed that, administering a CD39 inhibitor or blocking antibody reduced the tumor-induced suppression of CD4 and CD8 T-cell proliferation and enhanced the cytotoxic activity of cytotoxic T lymphocytes and NK cells. In a lung cancer model study, an anti-CD39 monoclonal antibody, which inhibits the mouse ectoenzyme CD39, was found to increase CD107a expression in infiltrating NK cells and stimulate IFN-γ release, leading to enhanced cancer cell killing and anti-metastatic effects cells [226]. Similarly, in mouse model of melanoma, the administration of anti-CD39 mAb stimulated the release of IFN-γ, resulting in the eradication of cancer cells [226]. A study by Lu et al. [227] demonstrated that the bifunctional antibody-ligand trap, ES014 (targeting CD39/TGF-β), effectively inhibited CD39, preventing the degradation of extracellular ATP, while also neutralizing autocrine/paracrine TGF-β near target cells leading to restoration of anti-tumor immunity. An experimental study by Jin et al. [228] found that the combination of tumor CD73 knockdown with tumor-specific T-cell transfer successfully cured all tumor-bearing mice. Notably, adoptive T-cell immunotherapy alone provided no therapeutic benefit in mice with tumors that did not undergo CD73 knockdown. Another study in tumor-bearing mouse models, showed that anti-CD73 antibodies can amplify the anti-cancer effects of both anti-CTLA-4 and anti-PD-1 immunotherapies in MC38-OVA (colon) and RM-1 (prostate) subcutaneous tumors and established metastatic 4T1.2 breast cancer. The activity of anti-PD-1 mAb was also enhanced by anti-CD73 mAb, against 3-methylcholanthrene-induced fibrosarcomas [229]. In a study conducted by Perrot et al. [230], it was observed that the antibodies IPH5201 and IPH5301, which target the human membrane-associated and soluble forms of CD39 and CD73 respectively, effectively inhibited the hydrolysis of immunogenic ATP into immunosuppressive adenosine. The mechanism of action involved stimulating dendritic cells and macrophages, as well as restoring the activation of T cells isolated from cancer patients. Drugs that block the A2AR-mediated adenosinergic pathway could boost anti-tumor immunity by counteracting the effects of extracellular adenosine generated by both tissue cells and Tregs. Pharmacological treatment of mice with A2AR antagonists enhanced anti-tumor T-cell activity, leading to greater inhibition of tumor growth, destruction of metastases, and reduced neovascularization of cancerous tissues [230]. Current clinical trials are evaluating adenosinergic pathway targets either as monotherapy or in combination therapy (Table 10).

Table 10.

Clinical trials evaluating adenosinergic pathway targets

Clinical Trial ID	Intervention against OX40	Cancer type	Status	Phase
NCT05508373	JS019, a mAb targeting CD39	Advanced solid tumors	Active	I
NCT05234853	PUR001, an anti-CD39 mAb	Advanced solid tumors	Active	I
NCT04261075	IPH5201, a CD39-blocking mAb±durvalumab (anti-PD-L1)±oleclumab (anti-CD73)	Advanced solid tumors	Active	I
NCT05075564	ES002023, an anti-CD39 mAb	Advanced or metastatic solid tumors	Active	I
NCT05381935	ES014, an anti-CD39/TGF-β bispecific antibody	Advanced or metastatic solid tumors	Withdrawn	I
NCT04336098	SRF617 anti-CD39 mAb with pembrolizumab, gemcitabine, albumin bound paclitaxel	Advanced solid tumors	31% patients experienced disease stabilization at 8 weeks	I
NCT04306900	TTX-030, anti-CD39 antibody with immunotherapy± chemotherapy	Advanced solid tumors	Active	Ib
NCT05431270	PT199, an anti-CD73 mAb	Advanced solid tumors	Active	I
NCT05174585	JAB-BX102, an anti-CD73 mAb± pembrolizumab	Advanced solid tumors	Active	I/IIa
NCT04797468	HLX23, a CD73 inhibitor	Metastatic solid tumors	Withdrawn prior to enrollment, need to reevaluate the study design	-
NCT04104672	Quemliclustat (AB680), a small-molecule CD73 inhibitor	Advanced pancreatic cancer	Safe and tolerable, 37% reduction in the risk of death and a 5.9-month improvement in median overall survival	I/Ib
NCT03736473	MEDI9447 (oleclumab), anti-CD73 mAb	Advanced solid malignancies	All six patients reported adverse events and disease progression at the first on-treatment evaluation	I
NCT04148937	LY3475070, a CD73 inhibitor±pembrolizumab	Advanced solid malignancies	Initial findings: manageable safety profile, preliminary antitumor activity has been observed	I
NCT03549000	NZV930 (CD73 targeting mAb)±PDR001 and/or NIR178 (adenosine A2a receptor antagonist)	Advanced malignancies	Terminated	I/Ib
NCT02754141	BMS-986179 (CD73 mAb)±nivolumab	Advanced solid tumors	Well tolerated, complete and persistent CD73 target engagement	I/IIa
NCT05227144	ORIC-533, an oral small-molecule inhibitor of CD73	Relapsed or refractory multiple myeloma	Increases in circulating NK and CD8+ T cells were noted after one treatment cycle, with enhanced CD8+ T-cell activation at higher dose levels	Ib
NCT05174585	JAB-BX102 (CD73 mAb)±pembrolizumab	Advanced solid tumors	Active	I/IIa
NCT05431270	PT199, an anti-CD73 mAb	Advanced solid tumors	Active	I
NCT03549000	NZV930, an anti-CD73 mAb±PDR001 (spartalizumab, an anti-PD-1 antibody)±NIR178 (taminadenant, an adenosine 2A receptor antagonist)	Advanced malignancies	Specific efficacy outcomes not known yet	I/Ib
NCT04672434	Sym024 (S095024), an anti-CD73 mAb±Sym021 (an anti-PD-1 antibody)	Advanced solid tumor malignancies	TEA showed a sustained decrease in free soluble CD73 in blood at doses ≥1,500 mg and dose-dependent CD73 modulation, with >80% inhibition achieved	I
NCT05143970 (CHANCES study)	IPH5301, an anti-CD73 mAb±chemotherapy and trastuzumab	Advanced solid tumors	Preliminary signals of antitumor activity	I
NCT04572152	AK119, an anti-CD73 mAb,+AK104 (cadonilimab), a bispecific Ab targeting PD-1 and CTLA-4	Advanced or metastatic solid tumors	Early signs of anti-tumor activity were observed across multiple tumor types	I
NCT05119998	IBI325, an anti-CD73 Mab±sintilimab, an anti-PD-1 Ab	Advanced solid tumors	Active	Ia/Ib
NCT04940286	Oleclumab (an anti-CD73 Ab), gemcitabine, nab-paclitaxel, and durvalumab (an anti-PD-L1 Ab),	Pancreatic cancer	Active	II
NCT03954704	Dalutrafusp alfa (formerly GS-1423) a bifunctional Ab targeting CD73 and mFOLFOX6 (TGF-β)	Advanced solid tumors	Complete CD73 target occupancy on B cells and CD8+ T cells was observed	I
NCT04148937	LY3475070 (CD73 inhibitor)+pembrolizumab	Advanced solid tumors	Active	I
NCT02754141	BMS-986179 (an anti-CD73 Ab)+nivolumab	Advanced solid tumors	Complete and persistent CD73 target engagement in both tumor tissues and peripheral blood	I/IIa
NCT04989387	INCA00186, (anti-CD73 mAb±retifanlimab±INCB106385	Advanced solid tumors	Active	I
NCT05501054	Ciforadenant (A2AR antagonist)+ipilimumab+nivolumab	Advanced renal cell carcinoma	Active	Ib/2
NCT04969315 (ADPORT-601)	TT-10 (PORT-6), an A2AR antagonist	Advanced selected solid tumors	Active, not recruiting	I/II
NCT03207867	Taminadenant (NIR178), an A2AR antagonist	Advanced solid tumors and NHL	Terminated	II
NCT02403193	Taminadenant (formerly PBF-509/NIR178)±spartalizumab (PDR001), an anti-PD-1 Ab	Advanced NSCLC	Monotherapy: one CR and seven cases SD Combination therapy: one partial PR and 14 cases of SD	I/Ib
NCT05117177	Inupadenant (EOS100850), an A2AR antagonist	Advanced solid tumors	Enhances T-cell and B-cell immune responses and promotes tertiary lymphoid structure maturation within tumors	I
NCT04580485	INCB106385, A2AR antagonist±retifanlimab (INCMGA00012)	Advanced solid tumors	Active	I
NCT04262856 (ARC-7 study)	Zimberelimab (anti-PD-1)±domvanalimab (AB154), anti-TIGIT±etrumadenant (AB928), A2a/b adenosine receptor antagonist	Metastatic NSCLC.	Interim analysis: clinically meaningful improvements	II
NCT05177770	Etrumadenant±SRF617 (CD39 inhibitor)±zimberelimab (AB122)	mCRPC	Terminated	II
NCT04089553	AZD4635 (A2AR) with durvalumab or oleclumab	mCRPC	Minimal antitumor activity with a manageable safety profile	II
NCT03454451	CPI-006 (mupadolimab), a CD73 mAb) with ciforadenant or pembrolizumab	Advanced cancers	Early evidence of anti-tumor activity was noted with CPI-006 monotherapy	I/1b
NCT05403385 (A2A-005 study)	Inupadenant (EOS100850), small-molecule A2AR antagonist +carboplatin+pemetrexed	Stage III+Stage IV non-squamous NSCLC	ORR was 63.9% across all patients	II
NCT04580485	INCB106385+immunotherapy	Advanced solid tumors	Dose-escalation phase has been completed, and the study is currently in the dose-expansion phase	I
NCT05501054	Ciforadenant (CPI-444)+ipilimumab+nivolumab	Advanced RCC	Active	Ib/II
NCT03549000	NZV930 (anti-CD73 mAb)±PDR001±NIR178	Advanced malignancies	Terminated	I/Ib
NCT02403193 (AdenONCO study)	PBF-509 (taminadenant/NIR178), A2AR antagonist,±PDR001 (spartalizumab), an anti-PD-1	Advanced NSCLC	Objective responses were observed in both treatment groups, stable disease was achieved in several patients	I/Ib
NCT04895748	Taminadenant±DFF332, targeting hypoxia-inducible factor 2 alpha (HIF-2α)±everolimus (RAD001), mTOR inhibitor± spartalizumab (PDR001), anti-PD-1		Favorable safety profile and preliminary anti-tumor activity, however study was halted	I/Ib

TEA, target engagement assessments; NHL, non-Hodgkin lymphoma; NSCLC, non-small cell lung cancer; CR, complete response; SD, stable disease; PR, partial response; A2AR antagonist, adenosine A2A receptor antagonist; mCRPC, metastatic castration-resistant prostate cancer; ORR, overall response rate; RCC, renal cell carcinoma; HIF-2α, hypoxia-inducible factor 2 alpha; mTOR, mechanistic target of rapamycin.

TAM receptors (Tyro3, AXL, and Mer)

TAM receptors (Tyro3, AXL, and Mer) are a family of receptor tyrosine kinases expressed on various immune and tumor cells. Structurally, they consist of an extracellular domain, a transmembrane domain, and a conserved intracellular kinase domain [231]. These receptors, along with their vitamin K-dependent ligands Gas6 and protein S, are critical in apoptotic cell clearance, immune modulation, and cancer progression. They regulate tumor growth, survival, invasion, and metastasis while also facilitating immune evasion. Among them, AXL is particularly associated with aggressive tumor phenotypes and therapy resistance, making it a key target for cancer treatment [231,232].

In preclinical models, inhibitors specifically targeting AXL have demonstrated efficacy in inhibiting tumor growth and metastasis, as well as in enhancing anti-tumor immune responses by reprogramming the TME towards a more immune-supportive state [232]. In research conducted by Lin et al. [233], both genetic and pharmacological inhibition of AXL in resistant models led to a reduction in cell proliferation, migration, invasion, and tumor growth. These effects were notably enhanced when AXL inhibition was paired with docetaxel treatment. In a study by Taniguchi et al. [234], AXL inhibition decreased the viability of EGFR-mutated lung cancer cells overexpressing AXL treated with osimertinib (EGFR-tyrosine kinase inhibitor), resulting in reduced tumor size and delayed tumor regrowth compared to treatment with osimertinib alone. In a preclinical study, glioblastoma cell lines U118MG and SF126, were treated with temozolomide and radiation, with and without AXL tyrosine kinase inhibitor. In the group treated with temozolomide and radiotherapy along with AXL tyrosine kinase inhibitor (R428) showed significantly increased therapeutic effects [235]. Promising preclinical findings have prompted the initiation of clinical trials aimed at evaluating the safety, efficacy, and therapeutic potential of Axl inhibitors, both as monotherapy and in combination with other treatment modalities (Table 11).

Table 11.

Clinical trials evaluating AXL inhibitor

Clinical Trial ID	Intervention against OX40	Cancer type	Status	Phase
NCT03990454	SLC-391, receptor tyrosine kinase AXL inhibitor	Advanced solid tumors	Results awaited	I
NCT02988817	Enapotamab vedotin, AXL ADC (HuMax-AXL-ADC)	Specified solid tumors	Well-tolerated, encouraging preliminary anti-tumor activity	I/IIa
NCT04893551	Tilvestamab (BGB149), AXL mAb	Relapsed, platinum-resistant, HGSOC	Terminated	Ib
NCT02729298	TP-0903 (dubermatinib), an oral AXL kinase inhibitor	Advanced solid tumors	Well-tolerated, preliminary evidence of clinical activity	Ia/Ib
NCT02424617	Bemcentinib (BGB324), a selective AXL kinase inhibitor+erlotinib, EGFR inhibitor	IIIb or Stage IV NSCLC	Preliminary: overall disease control rate of 33%	I/II
NCT02922777 (BGBIL005)	Bemcentinib+docetaxel	Advanced NSCLC	Preliminary evidence of anti-tumor activity was observed	I/II
NCT03649321	Bemcentinib+Nab-paclitaxel, gemcitabine, cisplatin	Metastatic pancreatic adenocarcinoma	Active	I/II
NCT03184571 (BGBC008)	Bemcentinib+pembrolizumab	Advanced NSCLC	Generally well-tolerated, ORR of 33% and a CBR of 73%, who were composite AXL–positive	II
NCT02488408 (BGBC003)	Bemcentinib±low-dose cytarabine or decitabine	Acute myeloid leukemia or myelodysplastic syndrome	Survival benefit in newly diagnosed AML patients unfit for intensive chemotherapy	Ib/II
NCT03255083	DS-1205c, an oral AXL inhibitor, +osimertinib (EGFR-tyrosine kinase inhibitor)	Metastatic or unresectable EGFR-mutant NSCLC	Stable disease was achieved in approximately 69% of patients. No complete or partial responses were observed	I
NCT03599518	DS-1205+gefitinib	Metastatic or unresectable EGFR-mutant NSCLC	No patients achieved a complete or partial response. Stable disease was observed in 25% of patients	I

AXL, AXL receptor tyrosine kinase; ADC, antibody-drug conjugates; HGSOC, high-grade serous ovarian; EGFR, epidermal growth factor receptor; NSCLC, non-small cell lung cancer; ORR, overall response rate; CBR, clinical benefit rate.

CONCLUSION AND FUTURE RESEARCH DIRECTIONS

To overcome the limitations of PD-1/PD-L1 inhibitors, novel immune checkpoint targets such as TIGIT, LAG-3, and VISTA have emerged as promising therapeutic strategies. Combination therapy approaches, particularly those integrating ICIs with co-stimulatory agonists, are gaining traction as a means to enhance anti-tumor immunity and combat resistance. Similarly, the engagement of co-stimulatory molecules such as ICOS, OX40, and CD137 holds potential to enhance immune responses by stimulating T cell activation and function. Additionally, targeting TME components, including the CD73 adenosinergic pathway and TAM receptors (Tyro3, AXL, and Mer), may further disrupt immunosuppressive mechanisms, creating a more favorable landscape for immune-mediated tumor destruction. Despite these advances, several key research directions must be explored.

Biomarker-driven patient stratification

The identification of predictive biomarkers for novel immune targets remains critical for optimizing patient selection and treatment efficacy. Future studies should focus on developing robust biomarker panels that guide personalized immunotherapy approaches.

Combination strategies to overcome resistance

While ICIs have revolutionized cancer therapy, resistance mechanisms continue to limit their long-term efficacy. Investigating rational combinations of immune modulators, chemotherapy, radiotherapy, and targeted therapies will be essential to overcoming resistance and expanding treatment benefits to a broader patient population.

Personalized neoantigen-based immunotherapies

Advances in genomics and proteomics have enabled the identification of tumor-specific neoantigens. Future research should explore personalized neoantigen-based cancer vaccines and adoptive T cell therapies to enhance tumor specificity while minimizing off-target effects.

Integration of AI in immunotherapy research

Artificial intelligence (AI)-driven multi-omics analysis and deep learning models can facilitate the discovery of novel immune targets, predict patient responses, and optimize treatment regimens. Future studies should explore AI applications in immunotherapy design, biomarker discovery, and clinical trial optimization.

Role of extracellular vesicles in immunomodulation

Emerging evidence suggests that extracellular vesicles mediate immune cell communication and modulate immune responses within the TME. Investigating their role in immunotherapy resistance and their potential as therapeutic vectors could open new avenues for treatment innovation.

Next-generation immune checkpoints and co-stimulatory molecules

Ongoing efforts should aim to identify and characterize novel immune checkpoints and co-stimulatory pathways beyond the currently known molecules. Understanding their mechanisms of action and therapeutic potential will be crucial for expanding the immunotherapy landscape.

Long-term immunological memory and safety considerations

The durability of immune responses and potential long-term immune-related adverse events remain important considerations in immunotherapy development. Future research should focus on strategies to enhance long-term immunological memory while minimizing immune toxicity. By addressing these research directions, future immunotherapies can be more effective, personalized, and accessible, ultimately transforming cancer treatment paradigms and improving patient outcomes.