Since its emergence, immunotherapy, as a rapidly evolving area in the field of oncology, has become a prominent and promising means of therapy in various types of cancer. Whilst encompassing a broad range of treatment agents such as immune checkpoint blockade therapies, vaccines, and adoptive cell therapies, strategies largely focused on harnessing the body’s immune system by modulating coinhibitory and costimulatory receptors expressed on immune cells (1). These coinhibitory and costimulatory receptors, or immune checkpoints, are primarily expressed on T cells and function in regulating and finetuning the body’s immune response when stimulated (1). Thus, their vital role in modulating the immune system and the ability of cancer cells to alter the function and expression of these receptors make them attractive therapeutic targets. With the majority of approved immune checkpoint therapies directed towards blocking coinhibitory receptors such as cytotoxic T lymphocyte-associated molecule-4 (CTLA-4), programmed cell death receptor-1 (PD-1), and programmed cell death ligand-1(PD-L1) to revitalize the immune response against tumors, there remains room for the development, improvement, and clinical application of therapeutics that target costimulatory receptors capable of enhancing the immune response (1, 2). Here, we seek to review one of the costimulatory receptors, 4-1BB (CD137/ILA/TNFRSF9), and the progress made, trends, and future outlooks in the development of therapeutics that target it.

Stimulation of 4-1BB, a surface receptor expressed on activated T cells amongst many other types of immune cells, activates various signaling pathways involved in the generation of a potent cellular immune response. Thus, its ability to modulate a wide range of immune cells and, in particular, its role in mediating T cell survival, proliferation, and effector function, have made it an attractive target, amongst other costimulatory molecules such as OX40, GITR, and CD27, in the development of cancer immunotherapy (3, 4). This clinical promise and value of 4-1BB are reflected in the continuous advancements and growth in the knowledge of the receptor as well as the breadth of therapeutic strategies that have gone through or are currently undergoing pre-clinical and clinical trials. Thus, we provide a comprehensive review and a greatly needed update of the receptor, 4-1BB, and its role in disease and immunotherapy. We will discuss the recent progresses that have been made and the current trends in the development of therapeutics targeting the receptor, concluding with potential strategies and outlooks for future research.

Recent studies have indicated the regulatory role of posttranslational modifications (PTMs), specifically glycosylation and ubiquitination, in 4-1BB expression (52, 53). Glycosylation, the covalent conjugation of carbohydrate chains, at two different sites (N138 and N149) of the 4-1BB protein was shown to have a role in the maturation and localization of the protein to the cell membrane, with its absence preventing 4-1BB membrane expression (53). Of note, glycosylation, including N-linked glycosylation, the covalent attachment of an N-acetylglucosamine (GlcNAc) to the nitrogen atom on the side chain of an asparagine (Asn/N) residue via an N-glycosidic bond, has been shown to have a contributory role in the immune system, especially as means of intracellular regulation of immune receptor and ligand proteins (54).

With regard to 4-1BB, its two N-linked glycosylation sites (N138, N149) were determined to be located away from the receptor-ligand interface, on N-X-S/T motifs, the motif where canonical N-glycosylation occurs, in CRD4, suggesting that it did not function in mediating the 4-1BB/4-1BBL interaction ( Figure 2 ) (25, 54). Despite having been long known to be heavily modified by glycosylation, little was known about the biological importance of this modification on the receptor. However, recently, Sun et al. elucidated the role of this glycosylation, determining that it facilitates 4-1BB membrane localization by preventing its multimerization intracellularly and, thus, permitting its membrane transportation and fast turnover (53). With the absence of the N-glycans, the “under-glycosylated” proteins form stable dimers and oligomers, which, consequently, hinders the trafficking of the proteins to the cell membrane (53). Of note, glycosylation patterns have been observed to be altered in presence of disease, including cancer cells, which, consequently, impact the expression of proteins, including cell membrane receptors (55). This difference in glycosylation moieties and profiles may, perhaps, serve as a basis for future therapeutic development.

Furthermore, in terms of ubiquitination, 4-1BB has been found to have four different sites of ubiquitination on its intracellular domain (K214, K218, K219, K225) ( Figure 2 ) (52). Sun et al., thus, derived that 4-1BB is degraded and cleared via the ubiquitin-proteosome system (UPS) and such degradation can be slowed with the administration of a proteosome inhibitor or insertion of mutations at the ubiquitination site residues (52). The prolonged expression of 4-1BB at the cell surface, mediated by these strategies, allowed for augmented signaling down the receptor and transcription of the downstream genes. In addition, N-glycosylation of mature 4-1BB was observed to cause the receptor to be more prone to ubiquitination-mediated degradation, suggesting an interplay between the glycosylation and ubiquitination modifications of the protein (52). This suggests room for clinical intervention, such as treatment with proteasome inhibitors or inhibitors of enzymes involved in the ubiquitination and degradation processes, to prolong 4-1BB expression and signaling. Altogether, these findings indicate the potential of the glycosylation or ubiquitination status of 4-1BB as predictors of response to 4-1BB targeted therapy as well as their modulation as a clinical strategy in the treatment of diseases.

With its broad expression profile, 4-1BB signaling have been shown to have a significant role in the body’s immune modulation. This was demonstrated in studies by Kwon et al. where T cells of 4-1BB deficient mice maintained their proliferative ability in response to stimulation, yet failed to secrete cytokines and exert their cytotoxic T lymphocyte activities (56). Thus, with its expression in diverse cell types and biological significance, 4-1BB has further been shown to be involved in a wide variety of pathologies. Although largely known as a costimulatory molecule functioning in the activation of the immune response, 4-1BB signaling was also observed to be involved in its modulation, as in autoimmune diseases, as well as in driving diseases, as in cancer, depending on pathology, indicating that the essence of its activity may be cell-, tissue-, environment- dependent.

The anti-tumor activity of 4-1BB signaling has long been established. Melero et al. initially demonstrated the anti-tumoral potential of treatment with 4-1BB antibodies in murine P815 mastocytoma and Ag104A sarcoma, where it was displayed to be mediated by the amplified proliferation and effector functions of tumor-specific cytotoxic T cells induced by 4-1BB signaling upon antibody binding (57). Such anti-tumoral activity of 4-1BB was also detected in models of lymphoma and hepatocellular carcinoma (58, 59). Additionally, anti-tumoral activity of 4-1BB signaling was shown to be correlated with enhanced survival in a study done by Ju et al. where 4-1BB-intact mice demonstrated longer survival compared to 4-1BB-deficient mice in a melanoma model (60). Furthermore, loss-of-function mutations in the TNFRSF9 gene, which encodes for 4-1BB, have been reported to result in recurrent infections as well as an Epstein-Barr virus (EBV)-induced lymphoproliferation of both T and B cells (61, 62).

4-1BB signaling, as can be implied from its importance in modulating immunity, has been revealed to augment the body’s immune response against bacterial, fungal, and viral infections as well. Multiple studies have demonstrated that stimulating 4-1BB signaling results in the anti-pathogenic immune responses against infections, including those of Streptococcus pneumoniae, Listeria monocytogenes, Candida albicans, influenza virus, hepatitis C virus, and cytomegalovirus (63–67).

Of note, although known for its immunostimulatory effects as seen in the prior examples, 4-1BB signaling has also been shown to be involved in mitigating various autoimmune diseases. Agonistic 4-1BB antibody treatment demonstrated to be effective in alleviating experimental autoimmune encephalomyelitis, an experimental model of human multiple sclerosis, in which the increased secretion of IFN-γ by CD8⁺ T cells, in turn, induced the expression of indoleamine-2,3-dioxygenase (IDO), an immunosuppressive immune regulator, on APCs (68). A similar mechanism against autoimmunity was also observed in models of rheumatoid arthritis (69). In addition, 4-1BB signaling exhibited protective activities against systemic lupus erythematosus-like autoimmune disease in mice, with its deletion exacerbating the disease (70). Stimulation of 4-1BB signaling mediated by a 4-1BB antibody has also been reported to exert preventative effects on the development of autoimmune type 1 diabetes in non-obese diabetic (NOD) mice (71).

As a prominent mediator of immune responses expressed on various cell types, 4-1BB signaling not only exerts protective effects, but is also capable of driving pathologies such as the adverse effects observed following administration of therapeutic 4-1BB antibodies. The persistent stimulation of 4-1BB signaling and, consequently, continuous activation of T cells have been shown to result in granuloma formation in tumor-draining lymph nodes due to the excessive recruitment of macrophages (72). Furthermore, liver-associated toxicity has been reported to be a common problem associated with therapeutic 4-1BB antibody treatment. Dubrot et al. showed that 4-1BB antibody treatment results in CD8⁺ T cell infiltration into the liver causing inflammation and increased transaminase expression (73). Such infiltration, however, was not associated with clinical benefit in the setting of tumors in or around the liver tissue (73). In addition, activation of liver-resident myeloid cells through the 4-1BB signaling pathway has been shown to cause the production of IL-27, which is also associated with CD8⁺ T cell infiltration, increase in transaminase, and consequent hepatitis (74).

All in all, it can be concluded that 4-1BB expression and signaling has a significant role as a costimulatory immune receptor, whether it be therapeutic or pathogenic. Its wide expression profile and ability to harness the immune system mediated by its downstream signaling demonstrates the clinical promise, as well as challenges, it presents in the treatment of many diseases, including cancer. As can be implied, many efforts have been made in the development of immunotherapies targeting 4-1BB as well as in the strategies to avoid the off-target adverse effects and toxicities associated with 4-1BB stimulation.

The ability of 4-1BB signaling to induce a potent anti-tumoral immune response makes it a promising candidate in the development of cancer immunotherapies. As previously discussed, early studies of 4-1BB as a therapeutic target in cancers were performed by Melero et al. in 1997 where they demonstrated the clinical potential of 4-1BB agonism (57). Treatment with 4-1BB monoclonal antibodies was shown to be capable of eliciting profound tumor suppression mediated by the augmented T cell response subsequent of 4-1BB stimulation in both poorly- and highly- immunogenic murine tumor models (57). Many studies that followed further demonstrated the anti-tumor activity and promise of 4-1BB agonism, alone or in combination with other therapeutic strategies, in different types of cancer, including lymphoma, hepatocellular carcinoma, and melanoma (58, 59).

Briefly, the mechanism of 4-1BB agonism in the treatment of cancer is believed to be dependent on and mediated by the activation of multiple anti-tumoral immune pathways, primarily in CD8⁺ T cells that infiltrate the tumors. The agonistic antibodies are perceived to stimulate the CD8⁺ T cells and promote their proliferation, survival, and cytolytic activity including increased cytokine production, such as IFN-γ, ultimately leading to tumor regression (46). Of note, Martinez-Forero et al. determined that 4-1BB agonistic antibodies, upon binding to the receptor, induce 4-1BB and antibody/ligand complex internalization into an endosomal compartment where downstream signaling continues (75). Furthermore, it has been shown that secretion of perforin and granzymes and activation of the Fas/Fas Ligand (FasL) signaling pathway, a pathway that regulates apoptosis, are other potential cytolytic mechanisms of CD8⁺ T cells induced by 4-1BB agonism (76). 4-1BB stimulation with agonistic antibodies has also been observed to act on 4-1BB expressed on monocytes and macrophages where it induces metabolic and functional reprogramming of the respective cells, promoting their anti-tumor activities (77). In addition, it has been demonstrated that 4-1BB signaling also enhances the metabolic and mitochondrial functions of T cells, promoting their longevity and, consequently, a longer anti-tumor immune response following antibody treatment (78, 79).

Currently, there are two leading 4-1BB agonistic monoclonal antibodies (mAbs) under clinical investigation: urelumab (BMS-663513) and utomilumab (PF-05082566) (80, 81). Urelumab, a non-ligand-blocking fully human IgG4 mAb, was the first of the two to undergo clinical trials as monotherapy: a phase I/II open-label trial launched in 2005 studying urelumab in metastatic or locally advanced solid tumors (NCT00309023). Despite seeing an anti-tumor clinical response in patients, the therapeutic benefit of urelumab was dose-dependent and the drug was observed to have dose-limiting hepatotoxicity (80, 82). Utomilumab, a ligand-blocking fully human IgG2 mAb, on the other hand, demonstrated a more favorable safety profile in a phase I trial (NCT01307267) and was tolerated at higher doses while exerting relatively lower therapeutic efficacy as compared to urelumab (80, 81). Furthermore, Chin et al. observed that utomilumab required greater doses to achieve maximum activation of 4-1BB (23). Such differences in outcomes and dose requirements may, in part, be explained by the different binding properties of the two antibodies. Whereas urelumab binds to the N-terminus in CRD-1 of 4-1BB, utomilumab binds to the junction between CRD-3 and CRD-4, which is adjacent to the ligand binding site CRD2, and a part of CRD3 (23). Thus, while urelumab binds to 4-1BB independently of 4-1BBL, utomilumab binding has been shown to sterically occlude the ligand, which may explain its relatively lower efficacy. Additionally, the potent agonistic effects of urelumab could be explained by its ability to crosslink multiple receptor/ligand complexes by forming ternary complexes with 4-1BB and 4-1BBL which, in turn, enhances the activating signaling down the pathway (23). Nonetheless, due to the dose-limiting toxicities of urelumab and moderate efficacy of utomilumab, subsequent strategies largely focused on combination therapies that targeted 4-1BB along with other tumor-driving factors with the goal of drawing a potent anti-tumor response at a lower 4-1BB antibody dose, minimizing its associated adverse effects.

A common strategy studied in a variety of cancer types incorporated urelumab or utomilumab in combination with other conventional anti-tumoral therapeutics that have already been established, such as chemotherapy, radiotherapy, and other immunotherapies, with the goal of achieving synergistic or additive anti-tumor effects while minimizing unfavorable toxicities ( Table 1 ). Co-treatment of the 4-1BB antibody along with different classes of chemotherapy has been shown to elicit synergistic effects by preconditioning the immune environment, enhancing the proliferation of CD8⁺ cells following 4-1BB agonism, or by inducing 4-1BB expression (83–86). Treatment with a 4-1BB antibody in addition to radiotherapy has also been observed to evoke a strengthened anti-tumor immune response when compared to monotherapy of either regimen in different preclinical tumor models (87). Such synergy was explained, in part, to be mediated by the enhanced expression of 4-1BBL following radiotherapy, which augments immune signaling down the 4-1BB/4-1BBL pathway (87). This, in turn, enabled a potent anti-tumoral response at a lower dose of the 4-1BB antibody, limiting its associated hepatotoxicity. Immune checkpoint inhibitors are also another greatly studied drug class with synergistic potential in combination with 4-1BB agonism. Commonly observed antibodies in combination with urelumab or utomilumab include those specific to the immune checkpoint receptors CTLA-4 and PD-1 as well as the ligand for PD-1, PD-L1 ( Table 1 ). The agonistic effect on a costimulatory immune receptor and antagonistic effect on a coinhibitory immune receptor induced by the dual antibody treatment were observed to have synergistic anti-tumoral outcomes mediated by the enhanced effector functions and tumor infiltration of CD4⁺ and CD8⁺ T cells (88, 89). Of note, 4-1BB stimulation has been shown to be associated with upregulated PD-1 expression, whereas PD-1/PDL1 signaling functions in tumor cell resistance to 4-1BB agonism therapy (90). This could explain, in part, the synergy observed in the combination therapy of PD-1 or PD-L1 antibodies with 4-1BB targeted therapy. In addition, combination therapy of a 4-1BB antibody with a CTLA-4 antibody was shown to be capable of ameliorating the autoimmune adverse effects, including inflammation in the liver, caused by the individual antibodies in a mouse model of colon cancer, suggesting the promise of dual treatment over monotherapy (91). Apart from dual therapy with coinhibitory receptor inhibitors, 4-1BB targeted antibodies have also been studied in combination with antibodies that bind to other costimulatory TNFRs, such as OX40. Likewise, coadministration of 4-1BB and OX40 antibodies resulted in synergistic effects in the proliferation and functions of CD8⁺ T cells, produced by the concurrent stimulation of two different receptors that modulate such anti-tumor immune responses in dichotomous, independent ways (92–94).

Other antibodies commonly studied in combination with 4-1BB antibodies, with the purpose of obtaining synergistic or enhanced anti-tumor responses, include those targeting epidermal growth factor receptors (EGFRs) or CD20. 4-1BB agonistic antibodies in combination with rituximab, a CD20 monoclonal antibody, have been studied in the treatment of lymphomas and leukemias where they demonstrated clinical benefit with 4-1BB stimulation on NK cells promoting their activation and expansion as well as the ADCC in response to rituximab administration (95). Similarly, 4-1BB agonism on NK cells and the resulting strengthened ADCC showed to augment the efficacy of EGFR- or human epidermal growth factor receptor 2 (HER2)- targeted antibodies, cetuximab and trastuzumab, respectively, in cancers that may have been refractory or unresponsive to monotherapy (96, 97).

Apart from the conventional forms of therapy that have been discussed thus far, there is a variety of different therapeutic strategies that are being applied in combination with 4-1BB agonism to enhance therapeutic efficacy and safety. There has been an emergence in the utilization of bi- and tri- specific antibodies as well as the application of adoptive cell therapy (ACT), such as CAR T cell therapy, with the aims of enhancing tumor specific activity and reducing systemic toxicity of 4-1BB stimulation ( Tables 1 , 2 ). The bi- and tri-specific antibodies commonly seen in studies are mainly composed of a 4-1BB binding fragment along with those that bind to tumor-associated receptors/antigens, primarily HER2 or PD-L1, that were discussed previously in the dual antibody regimens ( Figure 3 ). Such engineering of an antibody, so that it is capable of binding to both 4-1BB and another target that is known to be commonly upregulated in and associated with tumors, sought to increase specificity of the 4-1BB activation by localizing the agonistic antibody and the consequent immune stimulation to the tumor environment. This localization preserved the synergistic anti-tumor effect seen with dual therapy while reducing the adverse effects seen when 4-1BB is stimulated nonspecifically in the peripheral tissues outside the tumor. Results from preclinical studies of 4-1BB/HER2 or 4-1BB/PD-L1 bispecific antibodies reflected the aims of this design, restricting T cell stimulation to the tumor environments that are rich in HER2 or PD-L1, respectively, and reducing the toxicity caused when 4-1BB is activated in the periphery, including in the liver (98, 99). In terms of the trispecific antibodies that are currently undergoing clinical trials (CB307, NM21-1480), like the bispecifics, they are mainly designed to target 4-1BB along with another tumor-driving or associated factor. In addition to those binding domains, they are composed of an additional, third domain that binds to human serum albumin (HSA), which serves the purpose of prolonging the serum half-life of the antibodies (100). Even more, Compte et al. have developed a bispecific 4-1BB agonistic Fc-free trimerbody composed of three 4-1BB binding single-chain variable fragments (scFv) and three EGFR binding antibodies for use in cancer treatment (101). By removing the Fc region, the authors sought to enhance the relatively lower efficacy and mitigate off-target toxicities which they attributed to full-length IgG antibodies (101–103). The different takes in the approaches in the design and development of novel, non-canonical antibodies that optimize efficacy while minimizing toxicities are suggestive of the importance and promise of 4-1BB costimulation as a therapeutic strategy.

4-1BB targeting has also been studied in combination with ACTs, such as tumor infiltrating lymphocyte (TIL) infusion and CAR T cell therapy. The addition of a 4-1BB antibody to the adoptive transfer of TILs was observed to enhance TIL persistence as well as increase the effector/cytotoxic phenotype and function of CD8⁺ T cells in vitro, demonstrating clinical potential (104). Furthermore, in the context of CAR T cell therapy, incorporation of the 4-1BB intracellular domain into CAR T cells and signaling down the domain prevented the exhaustion of CAR T cells and prolonged their survival and expansion (105, 106). In a separate study involving a mouse model of B cell acute lymphoblastic leukemia, 4-1BB expressing CAR T cells also demonstrated enhanced anti-tumor activity with reduced adverse effects compared to CD28 expressing CAR T cells (107).

Overall, it appears that the current trend in the development of therapeutics that incorporate 4-1BB stimulating mechanisms focuses on bi- or tri-specific antibodies capable of targeting 4-1BB along with another tumor-driving factor. There is also an emergence of novel agonistic 4-1BB antibodies that are being studied in clinical trials such as ADG106 (Adagene) or EU101 (Eutilex), as monotherapy or in combination, that may perhaps have enhanced anti-tumor activities with reduced systemic toxicity as compared to urelumab or utomilumab ( Table 2 ). Nonetheless, it can be inferred that future therapeutic strategies will most likely be a form of combination therapy due to the toxicity associated with nonspecific 4-1BB stimulation and limited efficacy with monotherapy. Thus, it can be expected that there will be a continuous growth of multimodal therapeutics that target a tumor-associated factor that can synergize with 4-1BB stimulation as well as localize its effects to the tumor microenvironment. Apart from such strategies, another potential tactic may be targeting the PTMs on 4-1BB. As previously discussed, the glycosylation and ubiquitination PTMs were shown to have a prominent effect on 4-1BB expression and signaling and, thus, perhaps may serve as potential targets in enhancing and prolonging 4-1BB expression on cells. In particular, Sun et al. identified the E3 ligase subunit involved in the ubiquitination and proteasomal degradation of 4-1BB, F-box/LRR-repeat protein 20 (FBXL20), which could be targeted in the development of therapeutics that enhance 4-1BB expression and signaling (52). Even more, based on the knowledge that glycosylation patterns vary in different cell types, screening for and targeting glycosylation moieties unique to cancer cells may be something that could be studied moving forward (55). All in all, it can be conjectured that future therapeutic regimens will focus and expand on the optimization of combination modalities for the treatment of different cancer types.

The 4-1BB receptor has long been established to be a prominent costimulatory receptor expressed on a wide variety of cell types, especially those of the immune system. This extensive expression profile, along with the role of the associated signaling pathways in harnessing a potent and durable immune response, makes the receptor an attractive target in the treatment of various diseases, including different types of cancer. The clinical potential and prominence of 4-1BB stimulation as a cancer treatment regimen is reflected in the numerous clinical trials that have been completed and are ongoing thus far. Nonetheless, the advantages of the receptor’s wide expression profile are accompanied by a critical limitation when a 4-1BB stimulating antibody is administered as monotherapy. The dose-limiting toxicities, especially hepatotoxicity, impose a limitation to the anti-tumor efficacy of the antibodies that stimulate 4-1BB. Thus, many emerging strategies sought to conserve the therapeutic potential of 4-1BB agonism while minimizing systemic toxicity in tissues outside the tumor. A strategy that has been predominantly applied in recent years and is expected to see an increase of is the utilization of bi- or tri- specific antibodies that enable the localization of 4-1BB stimulation to the tumor environment and elicit durable potent, and often synergistic, anti-tumor effects. Thus, drawing from the trends that we have summarized here, it can be projected that there will be a continuous emergence of strategies that utilize other tumor-specific targets in combination with 4-1BB agonism with the goal of localizing the immune stimulatory and anti-tumor effects of 4-1BB signaling to the tumor while minimizing off-target, peripheral 4-1BB activation. Nonetheless, there remains great room for the development and finetuning of novel strategies targeting 4-1BB as it indeed has a clinically significant and potent role in drawing durable and effective immune responses bolstered by its wide expression profile and associated signaling pathways.

Conceptualization and writing, AK. The literature search, MN. Conceptualization, review, editing, and supervision, S-OL. All authors contributed to the article and approved the submitted version.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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