Like CD28, CTLA-4 binds to the co-stimulatory molecules CD80 or CD86, but instead attenuates T cell activation (3). Therefore, CTLA-4 mainly exerts its immunosuppressive effect during activation in secondary lymphoid organs (Figure 2A) (4, 5). However, CTLA-4 signaling also enhances immunosuppression in other ways, e.g., by promoting the activity of regulatory T cells (Tregs) inside the tumor-microenvironment (6, 7), and the relative importance of these different mechanisms is not fully understood nor their contribution to therapy success. The anti-CTLA-4 monoclonal antibody ipilimumab (fully human IgG1, Bristol-Myers Squibb) increased the median overall survival (OS) among metastatic melanoma patients in a pooled analysis of phase II and III data and some patients even survived 10 years after treatment (8). However, 60% of the patients treated with anti-CTLA-4 experienced immune-related adverse events, which were severe (grade 3 or 4) in 10–15% of the patients (9).

Anti-CTLA-4 treatment has also been studied in non-melanoma tumors, demonstrating an 8% partial response rate in patients with metastatic renal cell carcinoma (RCC) in a phase II study (10). Nevertheless, anti-CTLA-4 treatment is generally not effective in non-melanoma tumors, which might for instance be due to a low fraction of tumor-reactive T cells in these tumors, high expression of PD-L1, or the secretion of anti-inflammatory cytokines. Overall, long-term survival can be obtained with anti-CTLA-4 treatment in melanoma patients, but the toxicity is quite high and it is typically less effective in other tumor types.

Similar to CTLA-4, PD-1 signaling within secondary lymphoid organs impairs T cell priming and enhances differentiation into Tregs (11, 12). Moreover, because PD-L1 expression is frequently upregulated in cancers and tumor-infiltrating lymphocytes (TILs) can express high levels of PD-1 (4, 13), PD-1 inhibits T cell responses in the tumor. PD-1 can also be expressed on tumor cells, such as melanoma subpopulations, and contribute to tumorigenesis (14). Therefore, a part of the efficacy of anti-PD-1 therapy may be due to inhibition of this cell-intrinsic pathway active in some tumor cells (Figure 2B) (14). As expected from the predominantly peripheral role of PD-1, Pdcd1^−/− (the gene encoding for PD-1) mice developed mild, organ-specific autoimmune responses (15, 16). Large phase I studies led to the prompt approval of the anti-PD-1 monoclonal antibodies pembrolizumab (humanized IgG4, Merck) and nivolumab (fully human IgG4, Bristol-Myers Squibb, Ono Pharmaceuticals) for patients with unresectable or metastatic melanoma not responding to anti-CTLA-4 (17–19). Importantly, anti-PD-1 was superior to anti-CTLA-4 in the treatment of advanced melanoma in terms of progression-free survival (PFS; 47.3 versus 26.5%) (20). Because severe (grade 3–5) side effects also occurred less frequently in anti-PD-1-treated (13.3%) compared with anti-CTLA-4-treated patients (19.9%), anti-PD-1 treatment is currently the first-line treatment for unresectable or metastatic melanoma in the USA and the EU. In addition, the FDA approved anti-PD-1 for the treatment of Hodgkin lymphoma, non-small-cell lung carcinoma (NSCLC), RCC, and head and neck squamous cell carcinoma (HNSCC), because clinical trials demonstrated the safety and efficacy in these cancer types (21–26). Anti-PD-1 might also improve the treatment of bladder, gastric, ovarian, and triple negative breast cancer (4, 19). Furthermore, anti-PD-L1 (atezolizumab) was recently approved for the treatment of bladder cancer (urothelial carcinoma) (27). In summary, anti-PD-1 is less toxic yet more effective than anti-CTLA-4 and is also effective in the treatment of non-melanoma tumors.

Promising preclinical studies have shown that chemotherapy can indeed sensitize tumors to immune-checkpoint blockade by promoting T cell activation and infiltration into the tumor (33). Moreover, chemotherapy, such as by cisplatin, can also enhance responses to T cell based immune therapies by sensitizing the tumor cells to T cell-induced death rather than by ICD (34). For example, cisplatin has been shown to synergize with synthetic long peptide (SLP) vaccination and improve tumor-cell killing in a preclinical tumor model (35). With respect to the clinical application, chemotherapy (dacarbazine) combined with anti-CTLA-4 (ipilimumab) was first tested in metastatic melanoma patients. A phase II study showed that more patients responded to dacarbazine plus anti-CTLA-4 when compared to anti-CTLA-4 alone (14.3 versus 5.4%) (36). In addition, a phase III study demonstrated that this combination slightly increased the OS, when compared to dacarbazine alone (11.2 versus 9.1 months) (37) (note the difference between the latter two studies in terms of the monotherapy). However, immune-related severe adverse events (grade 3 or 4) also increased (56.3 versus 27.5%) (37).

Since chemotherapeutics promote antigen release and may promote the activation of tumor-specific T cells, combined chemotherapy and immune-checkpoint blockade may also be clinically effective in non-melanoma tumors. However, chemotherapy (paclitaxel and carboplatin) followed by anti-CTLA-4 (ipilimumab) resulted in only a minor increase of the immune-related PFS in a phase II and phase IIIb/IV study in NSCLC and extensive disease SCLC patients, compared with chemotherapy alone (38, 39). In conclusion, despite promising preclinical studies, combination therapies of immune-checkpoint blockade with chemotherapy have so far only slightly enhanced the clinical efficacy, while amplifying adverse effects. The lack of efficacy might be attributed to limited induction of ICD by chemotherapy. As ATP is thought to be important in the recruitment and activation of DCs during ICD, combinations of chemotherapy and inhibition of the ATP degrading enzyme CD39 followed by immune-checkpoint blockade might improve the clinical efficacy (40, 41).

Targeted therapies can also restore T cell activation, but subsequent expression of PD-L1 on tumor cells can lead to immune escape. For example, clinical data demonstrated that such escape occurred within 10–14 days after BRAF inhibition in tumors of patients with metastatic melanoma (42). Thus, combination approaches with immune-checkpoint blockade may function synergistically. However, a phase I trial combining BRAF^V600E inhibitor vemurafenib with anti-CTLA-4 (ipilimumab) in metastatic melanoma was halted due to severe hepatotoxicity (43). To date, such hepatoxity did not occur in an ongoing phase I trial with the BRAF^V600E inhibitor dabrafenib in combination with anti-CTLA-4 (ipilimumab) (44, 45), but clinical efficacy data are not yet available. Because PD-L1 is upregulated on tumor cells following BRAF inhibition, and anti-PD-1 therapy is less toxic compared to anti-CTLA-4, combining BRAF^V600E inhibitors with anti-PD-1 therapy seems promising. Indeed, preclinical data showed that the combination of anti-PD-1 or anti-PD-L1 with BRAF inhibition enhanced antitumor responses in a BRAF/PTEN inducible melanoma model (46), thus warranting clinical studies. Still, BRAF^V600E inhibition can potentially lead to activation of MAPK signaling in T cells, since wild-type BRAF is not inhibited. This may result in exhaustion or enhance immune suppression (47).

Until recently, the preclinical and clinical assessment of MEK inhibitors (e.g., tramatinib) in combination with immune-checkpoint blockade was limited, because MEK inhibitors disrupt T cell function transiently in vitro (48). Nevertheless, a synergistic effect of trametinib plus anti-PD-1, anti-PD-L1, or anti-CTLA-4 was observed in the preclinical CT26 carcinoma model (49). Triple therapy with MEK inhibitors, BRAF^V600E inhibitors, and anti-CTLA-4 may potentially optimize antitumor T cell responses with limited autoimmune side effects, since the additional MEK inhibitors might circumvent T cell hyper-activation caused by BRAF inhibition (47). Nevertheless, the combination of MEK inhibitors (trametinib), BRAF^V600E inhibitors (dabrafenib), and anti-CTLA-4 (ipilimumab) resulted in severe gastrointestinal toxicity in patients with metastatic melanoma in a phase I/II study (45). In contrast, MEK inhibitors (trametinib) and BRAF^V600E inhibitors (dabrafenib) have been successfully combined with anti-PD-L1 (durvalumab; MEDI4736) in melanoma patients in a phase I study (50). Thus, MEK or BRAF^V600E inhibitors might be safely and effectively used in combination with anti-PD-L1, but little clinical efficacy data are currently available.

Radiotherapy induces local tumor cell death, but can also modulate local and systemic immunity, in essence acting as an in situ vaccine. Radiotherapy can promote CD8⁺ T cell responses to radiotherapy-induced peptides (51), natural (52, 53) or exogenous tumor antigens (54, 55), or by promoting immune control within the tumor-microenvironment (51, 56–58) [reviewed in Ref. (59)]. CD8⁺ T cells can contribute to radiotherapy-induced reduction of the primary tumor (55), and radiotherapy can even induce regression of lesions that reside outside the field of radiotherapy, called the “abscopal effect.” However, this is a very rare phenomenon, reported 34 times since 1969 [reviewed in Ref. (60)], indicating that radiotherapy alone sub-optimally engages the cancer-immunity cycle (or does not overrule all the impediments that may still be there).

However, local and systemic responses may be achieved at the same time when radiotherapy is combined rationally with immunotherapy. Indeed, the Demaria/Formenti groups first demonstrated that radiotherapy combined with anti-CTLA-4 improved control of irradiated tumors and reduced non-irradiated lung metastases in preclinical models (61, 62). Similarly, PD-L1 or PD-1 blockade and radiotherapy-induced local combined responses in mouse models for colon carcinoma (MC38) and mammary tumors (4T1 and TUBO) (63), and resulted in long-term survival in a mouse model for intracranial gliomas (GL261) (64). In the latter case, long-term immunologic memory was achieved, because rechallenged, long-term surviving mice did not develop new tumors.

A number of clinical case studies showed that concurrent treatment with radiotherapy and anti-CTLA-4 (ipilimumab) promoted an abscopal effect in metastatic melanoma (3 × 9.5 Gy) as well as in NSCLC (5 × 6 Gy in 10 days) (65, 66). However, it is unclear whether this effect is caused by the combination of radiotherapy and anti-CTLA-4 (systemic effect of combination) or anti-CTLA-4 alone (systemic effect of immunotherapy), since the latter control was obviously not included in these case studies.

Confirming the challenge of obtaining systemic combined effects between radiotherapy and aCTLA-4 comes from a phase I/II study with castration-resistant prostate cancer (CRPC) patients, in which the tumors were first irradiated (1 × 8 Gy), followed by anti-CTLA-4 (ipilimumab) administration 2 days later in an attempt to maximize antigen presentation (67). In this study, relatively few severe adverse events (10%) were reported and a complete response occurred. However, no differences in median survival were found in a phase III trial among metastatic CRPC patients applying radiotherapy plus anti-CTLA-4 (ipilimumab) or placebo (11.2 versus 10.0 months) (68), again indicating that systemic combined effects between radiotherapy and anti-CTLA-4 are sub-optimal. Finally, a recent phase I study in metastatic melanoma patients reported an 18% abscopal response rate, but this is also expected from anti-CTLA-4 treatment alone (69). Anti-CTLA-4 treatment can result in the upregulation of tumoral PD-L1 expression (69). Indeed, the addition of anti-PD-1 or anti-PD-L1 to radiotherapy plus anti-CTLA-4 in a mouse model increased local combined responses and achieved cures in up to 80% of the animals, but combined effects on non-irradiated tumors were not assessed in this setting (69).

Hence, high (local) response rates are currently only achieved in combination with double immune-checkpoint blockade, and convincing clinical abscopal effects with radiotherapy and immune-checkpoint blockade are yet to be reported. In order to optimize the efficacy of combination approaches with radiotherapy, it is important to mechanistically understand how radiotherapy can optimally induce ICD to kickstart a new T cell response, which likely depends on the tumor type and location (hypoxic versus normoxic environment) and how irradiated and distal tumor-microenvironment are influenced by radiotherapy. Then, irradiation dose, scheduling, size of radiotherapy field (note: want this as small as possible to prevent damage to circulating lymphocytes that circulate through the RT field), timing, and delivery method can be optimized to achieve local and systemic therapeutic effects with immune-checkpoint inhibitors (70–72). For more details on preclinical and clinical efficacy of radiotherapy combined with aCTLA-4 see an elegant, recent review by Vanpouille-Box et al. (73).

In summary, the preclinical studies on combination approaches of immune-checkpoint blockade with stimulation of antigen release are promising, yet the clinical efficacy is currently limited. Furthermore, combination therapies with chemotherapy of BRAF-targeted therapies are potentially accompanied by severe side effects. Combination approaches with radiotherapy seem to have a more favorable toxicity profile, but, to date, local and abscopal antitumor responses are limited.

In a phase I study with advanced melanoma patients, vaccination with a modified sequence from glycoprotein 100 (gp100), a melanoma tumor antigen, increased the frequency of melanoma-specific CD8⁺ T cells (74). Nevertheless, the tumors progressed, implying that vaccination may benefit from combination with immune-checkpoint blockade. However, anti-CTLA-4 (ipilimumab) plus gp100 peptide vaccine did not improve the OS, compared with anti-CTLA-4 alone (10.0 versus 10.1 months) in a phase III trial in advanced melanoma patients (9). Thus, multiple antigens may need to be targeted to induce sufficient antitumor CD8⁺ T cell responses. Such a multi-peptide vaccine (gp100, MART-1, NY-ESO-1) increased the number of gp100⁺, MART-1⁺, and NY-ESO-1⁺ CD8⁺ T cells in advanced melanoma patients when combined with anti-PD-1 (nivolumab), and a promising 1-year survival of 87% was obtained in this phase I trial (75). Thus, early clinical data indicate that multi-peptide vaccines combined with immune-checkpoint blockade are highly promising.

Vaccination strategies in combination with immune-checkpoint blockade are also studied in non-melanoma tumors. Currently, a vaccine with a live-attenuated Listeria strain encoding the human papillomavirus (HPV) 16 oncoprotein E7 (ADXS11-001) in combination with anti-PD-L1 (durvalumab) is studied in a phase I/II trial in patients with cervical cancer or HPV-positive head and neck cancer (NCT02291055). Furthermore, a live-attenuated Listeria strain encoding for prostate-specific antigen level (PSA) (ADXS31-142) in combination with anti-PD-1 (pembrolizumab) is studied in a phase I/II trial in patients with prostate cancer (NCT02325557). In summary, combination approaches with live attenuated viruses are currently tested in the clinic, but clinical safety and efficacy data are not available yet.

Clinical trials combining checkpoint therapy with cellular vaccines are also ongoing, such as a phase I trial combining MART-1_26–35-loaded DC injection with anti-CTLA-4 (tremelimumab) in metastatic melanoma patients (76) and with anti-PD-1 (nivolumab) in patients with recurrent brain tumors (NCT02529072).

However, cellular vaccines consisting of irradiated GM-CSF-producing tumor cells (GVAX) may be more suitable, since they deliver multiple antigens. Granulocyte-macrophage colony-stimulating factor (GM-CSF) attracts DCs, stimulates the differentiation of monocytes into DCs, and amplifies DC cell maturation and antigen presentation (77, 78). Preclinical studies showed that subcutaneous injection of GVAX plus CTLA-4 blockade synergistically promoted tumor eradication in the B16 mouse melanoma model (response rate 80 versus 16% with GVAX alone) (79) due to an increase in CD8⁺ T cells and in effector T cell/Treg ratio in the tumor (80). GVAX plus PD-1 blockade also enhanced survival in the murine B16 melanoma, CT26 colon carcinoma, and pancreatic ductal adenocarcinoma models (81, 82). Furthermore, GVAX plus double checkpoint blockade (anti-CTLA-4 and anti-PD-1) enhanced tumor rejection to 100% in CT26 colon carcinoma and to 75% in ID8 ovarian carcinoma, compared to, respectively, 75 and 50% with double checkpoint blockade only (83). It should be noted that depigmentation was observed in 56% of the mice after combined GVAX and immune-checkpoint blockade (79), which correlated with antitumor activity. Clinical studies with cellular vaccines and checkpoint therapy are still rare. In a phase I/II trial with metastatic CRPC patients, treatment with GVAX plus anti-CTLA-4 (ipilimumab) was found to be safe and decreased the PSA by ≥50% in 25% of the patients (84). Thus, preclinical and early clinical data of cellular vaccines in combination with immune-checkpoint blockade are promising, warranting future clinical studies.

Granulocyte-macrophage colony-stimulating factor can also be directly administered instead of through production by irradiated tumor cells. In a phase II trial of systemic administration of recombinant GM-CSF (sargramostim) plus ipilimumab in metastatic melanoma patients, their 1-year survival increased upon combination therapy compared with anti-CTLA-4 alone (68.9 versus 52.9%) without toxicity differences (85). In addition, to minimize toxicity and optimize efficacy, GM-CSF can also be locally delivered in the tumor using modified oncolytic herpes viruses (talimogene laherperepvec; T-VEC) to produce GM-CSF. Such oncolytic viruses infect melanoma cells and then directly promote cell death with antigen release, stimulating antitumor immune responses (86). Intratumoral injection of T-VEC in melanoma patients in combination with ipilimumab treatment resulted in a response rate of 50% with a tolerable safety profile in a phase Ib trail. Currently, T-VEC combined with anti-CTLA-4 (ipilimumab) is studied in a phase II trial (NCT01740297) and combined with anti-PD-1 (pembrolizumab) in a phase Ib/III trail (NCT02263508), both in melanoma patients. The latter provided a clinical benefit and a phase III trial is planned (87, 88). Based on the clinical response rate of T-VEC and anti-CTLA-4, immune-checkpoint blockade with oncolytic viruses might be one of the most promising combination approaches.

CD40 is constitutively expressed on APCs and, like GM-CSF, agonistic antibodies for CD40 (anti-CD40) provoke DC maturation and the expression of co-stimulatory molecules. A 27.3% response rate was obtained with anti-CD40 (CP-870893) plus anti-CTLA-4 (tremelimumab) in a phase I study in melanoma patients (NCT01103635) (89). As anti-CD40 has been shown to upregulate PD-L1 in a mouse model (90), higher response rates might be obtained with anti-CD40 plus anti-PD-1. Indeed, anti-CD40 (APX005M) is currently studied in combination with anti-PD-1 (pembrolizumab) in a phase I/II trial in melanoma patients (NCT02706353). Furthermore, combination therapies with antagonistic antibodies for CD47, which provide “do not eat me” signals to APCs, might be used in the future in order to stimulate the engulfment of antigens (91). Thus, combination approaches with anti-CD40 or anti-CD47 can also be used to improve antigen presentation, but the available data show a smaller effect than other approaches to affect antigen presentation.

Overall, both the preclinical and initial clinical data for combination therapy with immune-checkpoint blockade and multi-peptide vaccines and oncolytic viruses are promising, whereas the combination with single peptide vaccines and anti-CD40 so far seem less effective.

Like immune-checkpoint blockade, agonistic antibodies for co-stimulatory molecules (e.g., CD27, CD137, GITR, and OX40) amplify T cell activation and, as a consequence, they may also enhance antitumor T cell responses. Various combinations with checkpoint blockade have been tested in mouse models (Table 2). The combination of OX40 and anti-CTLA-4 is synergistic in ovarian carcinoma (ID8), fibrosarcoma (MCA-205), and prostate cancer (TRAMP1) models (112, 113). Moreover, anti-GITR and anti-PD-1 have synergistic effects in the ID8 model (114). Some of these combinations have potentially less autoimmune side effects than double immune-checkpoint blockade while still having beneficial effects, for example, combination therapy involving anti-CD137 and CTLA-4 blockade in MC38 colon carcinoma tumors and GL261 glioblastoma (115, 116). In contrast, this combination treatment had no effect on B16 melanoma tumors (115, 116), which may be due to increased PD-L1 expression on those tumors.

Since PD-L1 expression can confer resistance to anti-CD137 treatment (122), combined CD137 activation and PD-1 rather than CTLA-4 blockade may further improve antitumor responses. Indeed, anti-CD137 plus anti-PD-1 resulted in increased efficacy in mouse models for colon carcinoma (ID8), melanoma (B16F10), and ovarian carcinoma (ID8) (117, 120, 121). Furthermore, triple therapy (activation of CD137 and blockade of PD-1 and CTLA-4) was even more effective in the ID8 model (117). However, CD137 agonists have been reported to induce severe liver inflammation (123, 124), and, therefore, more studies determining the toxic effects of combinations involving such agonists are needed. Moreover, intratumoral administration may help to minimize liver toxicity. Although liver inflammation was not detected in combination with anti-CTLA-4 or anti-PD-1 in preclinical studies (115, 117, 120), caution should be taken during its clinical assessment. Currently, a phase I study, in which the toxicity of anti-CD137 and anti-PD-1 is determined in patients with solid tumors, is in progress (NCT02179918). Combination approaches with anti-CD27 (Tnfrsf7) are also promising (125). The combination of anti-CD27 with anti-PD-1, but not anti-CTLA-4, eradicated tumors in a cervical cancer model (TC-1) (119). Currently, the combination of anti-CD27 (varlilumab) with anti-CTLA-4 (ipilimumab) or anti-PD-1 (nivolumab) is investigated in respectively patients with melanoma (NCT02413827) and solid tumors (NCT02335918) in phase I/II studies. Like the combination approaches with blockade of novel immune checkpoints, agonists for co-stimulatory molecules might have a more favorable safety profile, but caution is required with anti-CD137.

Summarizing the combinations with T cell stimulation, double immune-checkpoint blockade of CTLA-4 and PD-1 dramatically increased the response rate compared with monotherapy, but personalized treatment of anti-PD-1 with new immune-checkpoint inhibitors or agonists for co-stimulatory molecules can potentially minimize the severe side effects observed with CTLA-4/PD-1 combined blockade.

The growth factor VEGF inhibits T cell infiltration into the tumor by downregulation of adhesion molecules on endothelial cells (130). However, the immunosuppressive function of VEGF is much more diverse. For instance, VEGF also inhibits antigen presentation by DCs, enhances the expansion of Tregs, and mediates PD-1 upregulation on tumor-infiltrated T cells (131–133). High VEGF levels are associated with decreased OS in advanced melanoma patients after treatment with anti-CTLA-4 (134). Therefore, the combination of anti-VEGF-A (bevacizumab; humanized IgG1, Roche) and anti-CTLA-4 (ipilimumab) was investigated in a phase I trial in advanced melanoma patients. This combination resulted in a high CD8⁺ T cell infiltration and a median survival of 25.1 months (135), suggesting a substantial benefit compared to the 10.1 months median survival of anti-CTLA4 alone (9) (note though that this is another trial). In this trial, 28.3% of the patients experienced severe (grade 3 or 4) adverse events (135).

Another phase I trial applying a small molecule tyrosine kinase VEGF receptor (VEGFR) inhibitor (sunitinib) plus anti-CTLA-4 (tremelimumab) to patients with metastatic RCC resulted in acute renal failure, and the study was therefore halted (136). However, combined treatment with the generally safer anti-PD-1 and the VEGFR inhibitor sunitinib may be feasible. In preclinical studies, the combination of anti-VEGF-A and anti-PD-1 therapy was more effective than the monotherapies in a CT26 carcinoma mouse model (131). Moreover, preliminary clinical data of a phase I trial investigating the combination of anti-PD-1 (nivolumab) with either the VEGFR inhibitor sunitinib or with a multikinase VEGFR inhibitor (pazopanib) are promising (137, 138). In summary, combination therapies with anti-VEGF might provide a substantial survival benefit to patients, whereas caution is required with VEGFR inhibitor combination therapies due to renal toxicity.

Innate immune receptors (also known as pattern recognition receptors; PRR) such as TLR can detect a broad range of pathogen- and danger-associated patterns. Binding of these ligands to TLRs on DCs results in their maturation (139, 140). In addition, TLR9 agonists have been shown to increase T cell infiltration in the CT26 colon cancer model (141). Therefore, TLR9 agonists might be used to enhance T cell infiltration into the tumor. Currently, anti-CTLA-4 (ipilimumab) combined with a TLR9 agonist (MGN1703) is studied in a phase I trial in patients with advanced solid malignancies (NCT02668770). In summary, although clinical data are not yet available, combinations with innate immune receptor stimulation might improve T cell infiltration.

Epigenetic silencing of immune-related genes using hypermethylation contributes to immune evasion during tumor progression (142, 143). Hypomethylating agents, of which the nucleoside DNA methylation inhibitor Azacitidine (AZA) and 5-aza-2′-deoxycitidine (5AZA2) are most extensively studied, can be used to restore gene expression (144). 5AZA2 treatment has been shown to promote CD8⁺ T cell infiltration into the tumor in the EL4 lymphoma model, which was attributed to demethylation-induced CD80 expression on tumor cells (145). Therefore, hypomethylation can be used as a strategy to stimulate T cell infiltration into the tumor. Moreover, 5AZA2 treatment combined with anti-CTLA-4 is synergistic in murine mammary carcinoma TS/A and in mesothelioma AB1 models and results in high CD8⁺ and CD4⁺ T cell infiltration (146). Thus, clinical studies are needed as a next step to investigate the safety and efficacy of combination approaches with epigenetic modulation.

In conclusion, several approaches to stimulate T cell infiltration have been attempted but our knowledge on the efficacy of T cell infiltration stimulators combined with checkpoint blockade remains limited. This clearly is an area where more research is needed.

Adenosine inhibits T cell proliferation and cytotoxicity upon binding to the A2A receptor on T cells (155–157). In addition, adenosine can directly provoke metastasis upon interaction with the A2B receptor on tumor cells (158). As a consequence, the combination of an A2A receptor antagonist and anti-CTLA-4 or anti-PD-1 synergistically inhibited (metastatic) tumor growth in breast cancer (4T1) and melanoma (B16F10) preclinical models (159–161). Currently, the safety and tolerability of combined A2A receptor antagonist (CPI-444) and anti-PD-L1 (atezolizumab) treatment is studied in a phase I trial in advanced cancers (NCT02655822).

Adenosine is formed by dephosphorylation of adenosine monophosphate (AMP) by the ecto-enzyme CD73 (162). Thus, CD73 has an immunosuppressive and pro-metastatic effect. Moreover, CD73 stimulates angiogenesis through upregulated VEGF secretion by tumor cells (163). As a consequence, high expression of the enzyme CD73 is associated with a poor prognosis in various cancer types (164–166). This enzyme is also a potential biomarker for anti-PD-1 therapy, since CD73 expression limits anti-PD-1 efficacy (161, 167). Indeed, both combined treatment with anti-CD73 and anti-CTLA-4 or anti-PD-1 enhanced the antitumor activity in colon (MC38), prostate (RM-1), and metastatic breast cancer (4T1.2) models (168). Currently, the combination of anti-CD73 (MEDI9447) plus anti-PD-L1 (durvalumab) is investigated in the clinic in patients with advanced solid cancer (NCT02503774) in a phase I study. In summary, combination approaches with A2A receptor antagonists and anti-CD73 to improve tumor killing are currently studied in the clinic due to their success in preclinical studies.

Transforming growth factor-β can also contribute to immune suppression by stimulating Tregs (169). Preclinically, combined treatment with a TFG-β receptor kinase inhibitor I and anti-CTLA-4 synergistically inhibited primary and metastatic tumor growth in a melanoma model (BRAF^V600EPTEN^−/−) (170). The combination of the TFG-β receptor kinase inhibitor I (galunisertib) and anti-PD-L1 (durvalumab) or anti-PD-1 (nivolumab) are currently being clinically tested in patients with metastatic pancreatic cancer (NCT02734160, phase I) and NSCLC, hepatocellular carcinoma, or glioblastoma (NCT02423343, phase I/II). These clinical studies will demonstrate if combination approaches with TGF-β receptor kinase inhibitors may improve the treatment of various tumors.

In addition, TGF-β may impair radiotherapy-induced T cell priming, and TGF-β blocking antibodies can enhance T cell priming (step 2–3) and promote abscopal responses induced by (fractionated) radiotherapy in the 4T1 mouse model (53).

The presence of the tryptophan degrading enzyme IDO on APCs or tumor cells may also limit the killing efficacy of CTLA-4 or PD-1 blockade because tryptophan is required for T cell proliferation (171). Indeed, several preclinical studies demonstrated a survival benefit of combinations of anti-CTLA-4 or anti-PD-L1 with IDO inhibition by 1-methyl-d-tryptophan (1MT) treatment (Table 1). Thus, combination approaches with IDO inhibition may be efficacious, especially in patients with a high IDO expression. Finally, triple immune-checkpoint blockade of CTLA-4, PD-1, and IDO could further improve antitumor responses, but this approach probably does not increase the effectiveness compared to double checkpoint blockade in more established tumors (107).

Early clinical safety and efficacy data for the IDO1 inhibitor (epacadostat) in combination with anti-PD-1 (pembrolizumab) were promising for the treatment of various advanced cancers in a phase I/II study (e.g., melanoma, RCC, and NSCLC) (172). Currently, the IDO inhibitor epacadostat is also being tested in combination with anti-PD-1 (nivolumab; NCT02327078) and anti-PD-L1 (durvalumab; NCT02318277) for various cancers in phase I/II studies. In addition, 1MT (indoximod) plus anti-CTLA-4 (ipilimumab) or anti-PD-1 is being tested in melanoma patients (NCT02073123) in a phase I/II study. Thus, both preclinical and early clinical studies involving IDO inhibition have yielded promising results. However, because IDO inhibition seems particularly effective in tumors with a high IDO expression, it would be useful to target these patients specifically.

Summarizing the approaches that stimulate killing by T cells, various clinical trials currently investigate combination therapies that enhance such killing, but to date clinical safety and efficacy data are limited.