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24-07-2015 | Treatment | Article

Evolving synergistic combinations of targeted immunotherapies to combat cancer

Ignacio Melero, David M. Berman, M. Angela Aznar, José Luis Pérez Gracia, John Haanen


Immunotherapy has now been clinically validated as an effective treatment for many cancers. There is tremendous potential for synergistic combinations of immunotherapy agents and for combining immunotherapy agents with conventional cancer treatments. Clinical trials combining blockade of cytotoxic T lymphocyte-associated antigen 4 (CTLA4) and programmed cell death protein 1 (PD1) may serve as a paradigm to guide future approaches to immuno-oncology combination therapy. In this Review, we discuss progress in the synergistic design of immune-targeting combination therapies and highlight the challenges involved in tailoring such strategies to provide maximal benefit to patients.

Nat Rev Cancer 2015; 15: 457–472. doi: 10.1038/nrc3973

Subject terms: Cancer immunotherapy • Combination drug therapy • Immunotherapy

Key points

  • Clinical trials have validated immuno-oncology as a new pillar of anticancer therapy.
  • Combinations could involve two (or more) sequential or simultaneous immunotherapies, and/or immunotherapies in combination with conventional cancer therapies.
  • The programmed cell death protein 1 (PD1)–PD1 ligand 1 (PDL1) axis seems to be the most promising immuno-oncology target, and its blockade is likely to become the main foundation for combination strategies in the foreseeable future.
  • The paradigm of immuno-oncology combinations is to block PD1 and cytotoxic T lymphocyte-associated antigen 4 (CTLA4) simultaneously; this blockade is synergistic and shows clinical benefit in patients with melanoma but has an increased frequency of immune-mediated, albeit clinically manageable, toxic effects.
  • Even if designing rational combinations that provide optimal benefit to patients with cancer is a challenging process, there are a number of different combination immuno-oncology therapies currently in development.


Surface receptors of immune cells mediate intercellular communication and frequently operate in transient but well-structured cell–cell contacts known as immune synapses1 (Fig. 1). A major class of these receptors, checkpoint receptors2, inhibits the development or execution of effector functions of killer and pro-inflammatory lymphocytes. These mechanisms probably evolved to limit damage to non-infected cells in virally infected tissues and to attenuate excessive systemic inflammation and prevent the development of autoimmunity. However, these mechanisms are also being hijacked by tumours to avoid immune attack.

Figure 1: Receptor–ligand pairs of the immune system that are amenable to pharmacological manipulation with immunostimulatory monoclonal antibodies.

As well as mediating intercellular communication, surface receptors can also stimulate killer lymphocytes in coordination with clonally distributed antigen receptors (T cell receptors (TCRs)). Monoclonal antibodies (mAbs), through their bivalency and their ability to interact with receptors for their Fc fragments on neighbouring cells, can crosslink activating receptors and act as agonists3, 4, 5.

The approval of ipilimumab (a mAb that inhibits the checkpoint receptor cytotoxic T lymphocyte-associated antigen 4 (CTLA4)) for advanced-stage melanoma has validated the concept of using mAbs6 to activate antitumour immunity7, 8. Agonistic mAbs are also being developed for cancer immunotherapy and have shown some success in early-phase clinical trials.

In fact, multiple strategies for eliciting and enhancing antitumour immunity have been developed and are in the clinic9, 10 (Box 1). There is currently much focus on synergistically combining cancer immunotherapies to modulate immune outcomes. In pharmacology, the concept of synergy refers to a combination of agents having effects that are superior to what would be expected, in the dose–response curve, from adding their separate effects. Owing to mechanistic interactions, drugs with no single-agent activity can cooperate with other interventions to improve efficacy. In cancer immunotherapy, two parameters can help to define synergy: the intensity of the elicited measurable immune response against cancer; and the actual reduction (or disappearance) of tumour lesions.

Synergistic combinations of immunotherapies have largely been empirically developed by translating results from mouse models into clinical trials, the key example being the effective treatment of patients with melanoma by combining antibodies against the checkpoint receptors CTLA4 and programmed cell death protein 1 (PD1)11. Preclinical and clinical synergies have also been observed for immuno-oncology agents in combination with chemotherapy12, targeted therapies13, radiotherapy14, 15, anti-angiogenic agents16, 17 and partial surgical resections18. The field is developing rapidly, and the goal is to move from an era of empirical combinations to one of rational design by considering the compatibility of mechanisms that interact synergistically, either to mediate antitumour efficacy or to reduce on-target side effects19. In addition, tackling immune-escape strategies evolved by the tumour may necessitate adding agents to the combinations20. New combinations are being identified almost monthly, to the point that the colour-coding diagram included in various previous reviews6, 21 becomes almost instantly outdated6 (Fig. 2). The other field that is revolutionizing immunotherapy of cancer is adoptive T cell therapy22, 23, mainly in the form of T cells that are genetically modified to express chimeric antigen receptors against malignancies derived from transformed B cells, such as leukaemia and lymphoma23. Notably, there is preclinical evidence that adoptive T cell therapy and immunostimulatory mAbs can be synergistically combined24, 25. This Review summarizes the key elements and concepts of immunotherapy combinations and the challenges to the rational design of synergistic combination strategies involving immune modulators.

Figure 2: The rapid evolution of combination immunotherapy.

Targeting immunomodulatory pathways

The multiple natural negative feedback mechanisms that fine-tune the adaptive immune response, by activating or inhibiting T cell or natural killer (NK) cell function, provide pathways by which tumours can escape from the immune system. However, they also offer multiple opportunities for therapeutic intervention1, 9.

Inhibitory immune checkpoints

CTLA4 is expressed by activated T cells and regulatory T cells (TReg cells). Binding of CTLA4 to its ligands (B7-1 (also known as CD80) and B7-2 (also known as CD86)) on antigen-presenting cells (APCs) leads to inhibition of T cells26, 27. CTLA4-specific mAbs may bias the competition for ligands between CTLA4 and its co-stimulatory homologue, CD28, towards the latter. They may also function through other mechanisms, such as partially depleting TReg cells in the tumour microenvironment28, 29 and interfering with the CTLA4-mediated sequestration of co-stimulatory ligands at immune synapses30. As well as in metastatic melanoma7, 31, CTLA4 checkpoint inhibition with ipilimumab has confirmed clinical activity for other tumours32. Tremelimumab33 is another CTLA4-specific antibody that is in development for melanoma and other malignant diseases.

The receptor PD1 is expressed on activated T cells, B cells, NK cells and TReg cells34. PD1 has two identified ligands: PDL1 (also known as B7-H1; also has affinity for CD80 (Ref. 35)) and PDL2 (also known as B7-DC). PD1 signalling contributes to T cell exhaustion36, and tumours may exploit this pathway, as PDL1 is expressed on many solid tumours and is associated with a poor outcome37, 38, 39. Antibodies that inhibit the interaction between PD1 and its ligands have shown promising results in various tumour types. PD1-specific agents include nivolumab38, pembrolizumab40, 41, 42, 43 and pidilizumab (CT-011)44; PDL1-specific agents include atezolizumab (MPDL3280A)45, MEDI4736 ( identifiers NCT02087423 and NCT01693562) and MSB0010718C (NCT01772004, NCT01943461 and NCT02155647); and PDL2-specific agents include rHIgM12B7 (NCT00658892).

Lymphocyte activation gene 3 protein (LAG3; also known as CD223) and other inhibitory receptors are progressively expressed on T cells during exhaustion46, 47. Until recently, the only ligands identified for LAG3 were major histocompatibility complex (MHC) class II molecules, but evidence has shown that it can also be bound by galectin 3 with functional consequences48. The expression of LAG3 on tumour-infiltrating TReg cells and CTLs suggests that it may be involved in immune evasion by tumours; thus, blocking LAG3 may reverse T cell exhaustion and enhance antitumour immunity47, 49. BMS-986016, a LAG3-specific mAb, is in clinical development (NCT01968109 and NCT02061761).

T cell immunoglobulin and mucin domain-containing 3 (TIM3; also known as HAVCR2)50 is expressed on T helper 1 (TH1) cells and CTLs, but also on innate immune cells such as dendritic cells (DCs)51; its function differs depending on the cell type in which it is expressed. Expression of TIM3 by tumour-infiltrating lymphocytes (TILs) is common in melanoma52 and non-small-cell lung cancer (NSCLC)53, and it is thought to keep the lymphocyte status inactive or even to induce apoptosis upon ligation to galectin 9 or other, as yet undefined, ligands54. A functional interaction with carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) has also been described55.

Co-stimulatory receptors

CD137 (also known as 4-1BB and TNFRSF9) is a potent T cell and NK cell co-stimulatory receptor that is expressed at the cell surface following lymphocyte activation56. Promoting its signalling improves cytotoxic antitumour responses57 and T cell survival58, 59. Urelumab and PF-05082566 (Ref. 60) are agonistic CD137-specific mAbs that are under evaluation for various malignancies. Importantly, agonistic CD137-specific mAbs may enhance NK cell-mediated antibody-dependent cellular cytotoxicity (ADCC)61. Data from mouse models has shown that tumour cells coated with tumour-targeted mAbs induced the upregulation of CD137 expression on NK cells62, 63, 64. Subsequent addition of an agonistic CD137-specific mAb increased NK cell degranulation and tumour lysis. Therefore, preclinical evidence predicts powerful synergistic effects of CD137-specific mAbs when combined with rituximab (a mAb specific for CD20), trastuzumab (a mAb specific for human epidermal growth factor receptor 2 (HER2; also known as ERBB2)) or cetuximab (a mAb specific for epidermal growth factor receptor (EGFR))62, 63, 64.

Glucocorticoid-induced tumour necrosis factor receptor family-related protein (GITR; also known as TNFRSF18) is a co-stimulatory molecule that reverses TReg cell-mediated suppression of T cells and activates proliferation and effector functions in CD4+ and CD8+ T cells65, 66. Activating GITR may overcome self-tolerance, reverse TReg cell-mediated suppression and enhance antitumour immune responses67, 68, 69, 70. The agonistic GITR-specific mAbs TRX518 and MK-4166 are undergoing Phase I evaluation (NCT01239134 and NCT02132754).

OX40 (also known as TNFRSF4) is a co-stimulatory receptor expressed primarily on activated CD4+ and CD8+ T cells; it enhances antitumour immune responses by promoting T cell proliferation and survival71, 72, 73. OX40 agonists enhanced antitumour immunity by inhibiting TReg cells and promoting T cell survival in preclinical mouse models71, 72. Several agonistic OX40-specific mAbs are under clinical evaluation: namely, MEDI6469 (NCT01862900 and NCT01303705)74, MEDI6383 (NCT02221960), MEDI0562 (NCT02318394) and MOXR0916 (NCT02219724).

CD40 (also known as TNFRSF5) is a stimulatory surface receptor of the tumour necrosis factor receptor (TNFR) family; it promotes the activation of APCs and enhances their co-stimulatory and antigen presentation activities, leading to T cell activation75, 76. CD40 enables APCs to prime CD8+ T cells that then differentiate into CTLs. An agonistic CD40-specific antibody, CP870,893, is under evaluation77.

CD27 is a receptor of the TNFR superfamily that is expressed on resting and naive T lymphocytes but not on fully differentiated effector T cells. It is also expressed on a subset of NK cells78. Binding of CD27 to its ligand, CD70, on activated APCs enhances T cell activation, effector function, maturation, survival and long-term memory of the CD27-expressing cell. CD27 has a role in enhancing NK cell proliferation and cytotoxicity, and in B cell activation and immunoglobulin synthesis79, 80, 81, 82, 83, 84, 85. CDX-1127, a CD27-directed mAb, is under clinical development (NCT01460134).

Killer inhibitory receptors

Inhibitory killer cell immunoglobulin (Ig)-like receptors (KIRs) negatively regulate the cytotoxic activities of NK cells86 and some T cell subsets. KIRs recognize self-MHC class I molecules on cells and can inhibit NK cell activation. The loss or downregulation of self-MHC class I molecules (as occurs in most tumour cells) is sufficient to induce NK cell sensitivity: this is known as the 'missing self- recognition model' (Refs 87,88). However, tumour cells that retain proper MHC class I expression can evade immune surveillance by NK cells and thereby escape subsequent immune-mediated destruction. The use of KIR-specific mAbs for cancer therapy is under evaluation: lirilumab (also known as BMS-986015 and IPH2101) is a pan-specific KIR mAb designed to block multiple KIR family members89. NK cells and certain T lymphocyte subsets express other cytotoxicity-inhibiting receptors, such as NKG2A (also known as KLRC1)90 and CD96 (also known as TACTILE)91, which are also potential targets for cancer immunotherapy.

Combination immunotherapies for cancer

The breadth of immunotherapies in development provides multiple opportunities for combining — simultaneously or sequentially — therapies with distinct but potentially complementary mechanisms of action8, 32, 92, 93, 94, 95, 96, 97, 98, 99 (Fig. 3; Table 1). Successes of PD1 and PDL1 blockade in monotherapy regimens suggest that these agents will be preferred as the primary building blocks for combinations (Fig. 4). The discovery of synergistic combinations stems from careful preclinical appraisal100, 101 (Fig. 5a) and leads to stepwise testing of the simultaneous or sequential combinations in humans94, 99 (Fig. 5b,c,d).

Figure 3: Schematic representation of the main mechanisms of action postulated to mediate synergistic effects of combined immunotherapies.

Table 1: Outcomes from key clinical trials of immunotherapies in combination regimens

Figure 4: Building immunotherapy combinations on the pillar of PD1 or PDL1 blockade, and steps in the development of an immunotherapy combination.

Figure 5: Preclinical and clinical development of combinations of immunostimulatory monoclonal antibodies.

Combining immune checkpoint inhibitors

Combination therapy to block more than one immunomodulatory pathway may further enhance the antitumour efficacy of each individual treatment21.

The best-studied combination is that of CTLA4- specific and PD1-specific inhibitory mAbs, which have shown marked antitumour activity in many, but not all, tumour models102, 103, 104. These negative regulators affect different signalling pathways within T cells105, 106, suggesting that these therapeutic mAbs could synergize (for example, through enhancing CTL effector activity). Recent data have shown that ipilimumab treatment may lead to broadening of the melanoma-specific killer T cell response107, pointing towards a role in the priming of cancer-specific T cell immunity. In addition, evidence is emerging from mouse models showing that CTLA4-specific antibodies can promote the depletion of TReg cells specifically in tumours28, 29, 108, as well as repress TReg cell functions109. Interference with inhibitory pathways in effector T cells and the elimination of immune-suppressive cells such as TReg cells are currently thought to be the dominant mechanisms for enhanced antitumour activity by combined targeting of CTLA4 and PD1. However, the central role of inhibitory effects of PD1 expression in T cells that are chronically stimulated by antigen (along with the possible co-expression of CTLA4 on these cells) suggests that the combination has other possible mechanisms of action. Multiple repressive receptors such as CTLA4, TIM3 and LAG3 may cooperate with PD1 to maintain an exhausted phenotype of T cells, and thus combinations of blocking antibodies will be required to overcome this cooperation.

A Phase I clinical trial of ipilimumab and nivolumab in patients with metastatic melanoma94explored a dose escalation of nivolumab (starting at 0.3 mg per kg) added to the standard dose of ipilimumab (3 mg per kg once every 3 weeks for 4 doses). The maximum tolerated dose was declared at 3 mg per kg of each antibody, and impressive clinical responses were observed in patients treated with the lowest dose levels. In the combination group receiving nivolumab at a dose of 1 mg per kg and ipilimumab at a dose of 3 mg per kg, 40 patients were treated and 53% of these patients achieved objective responses, with 28% of patients showing an 80% or greater tumour reduction after the first scans (within 12 weeks)94. For patients who received concurrent ipilimumab and nivolumab across all doses, the 1-year and 2-year overall survival rates were notable at 85% and 79%, respectively94, suggesting that the activity of the combination was superior to that of either agent alone.

Combination therapy also led to an increase in the frequency of adverse events compared with prior experience with either antibody alone, in particular with nivolumab (for example, 61% of patients reported grade III adverse events in the group treated with nivolumab 1 mg per kg in combination with ipilimumab 3 mg per kg)94. Nevertheless, the variety of adverse events observed (including gastrointestinal effects such as diarrhoea and pathological inflammation of gut tissues) was within prior experience with these antibodies and consistent with a toxicology study of the combination in non-human primates (M. Selby, J. Engelhardt, L.-S. Lu, M. Quigley, C. Wang, B. Chen and A.J.K., unpublished observations). Interestingly, many of the grade III adverse events — in particular, those that led to establishing the maximum tolerated dose — were laboratory abnormalities without clinical correlates, such as asymptomatic elevation of lipase, amylase, alanine aminotransferase and/or aspartate aminotransferase.

Recently, the first results from a randomized, placebo-controlled Phase II study comparing ipilimumab plus nivolumab with ipilimumab alone were reported98. A total of 142 patients without prior systemic treatment were randomly assigned to the two groups, and results in patients with BRAF wild-type tumours (the largest group in this study) showed an objective response rate of 61% for the combination treatment and 11% for ipilimumab alone. Of the 72 patients with BRAF wild-type tumours assigned to the combination treatment, 22% experienced a complete remission, whereas there were no complete responders in the ipilimumab-alone arm. Clinical benefit in the combination arm was independent of PDL1 expression by tumour cells before treatment. By contrast, ipilimumab-induced responses were significantly more common in PDL1-positive tumours, similar to observations with PD1-specific therapy. The progression-free survival for patients treated with ipilimumab was 4.4 months and not yet reached for patients treated with ipilimumab plus nivolumab therapy (hazard ratio (HR) 0.40; P < 0.001). The adverse events observed in this trial were similar to those observed with the combination in the Phase I trial11. These efficacy and biomarker results have recently been confirmed in a randomized, three-arm Phase III clinical trial in which patients received ipilimumab, nivolumab or the combination regimen of ipilimumab plus nivolumab99. Although 945 patients were enrolled, the study was not statistically powered to compare progression-free survival and response rate between nivolumab alone and the combination. When the data are mature, comparative overall survival results from this clinical trial will be of paramount importance to demonstrate the full effect of combination immunotherapy. Adverse events of grade III or above were experienced by 68% of patients receiving the combination, compared with 43.5% and 55.6% of those receiving nivolumab alone and ipilimumab alone, respectively99.

On the basis of these extremely encouraging efficacy results, the combination of ipilimumab and nivolumab has been or is being explored in Phase III studies in renal cell carcinoma and NSCLC110, 111, and earlier-phase clinical trials are being conducted in various other tumour types (small-cell lung, triple-negative breast, pancreatic, gastric and bladder cancer). The combination of tremelimumab and a PDL1-specific antibody, MEDI4736, has also recently entered clinical testing (see Supplementary information S1 (table)).

PD1–PDL1 pathway blockade may be viewed as a pillar for future immunotherapy combinations (Fig. 4), and the promising results of combining ipilimumab and nivolumab have spurred the combinations of other immune checkpoint inhibitors preclinically and clinically6 (Fig. 2). Attractive combinations include PD1-specific antibodies with specific antibodies against LAG3 or TIM3, both of which have shown synergism with PD1 blockade in mouse tumour models49, 54. The rationale underlying synergistic effects is that each dysfunctional antitumour T cell could be kept unresponsive by more than one repressor and could only be fully capable to exert its functions when completely released from checkpoint inhibition112.

Combining checkpoint inhibitors and immunostimulatory mAbs

Another interesting combination approach involves delivering immunostimulatory mAbs with immune checkpoint inhibitors. These combinations pose new challenges for the clinical management of patients, and safety needs to be carefully addressed. A Phase I–II trial is underway to evaluate a combination of PD1-blocking agents with CD137-specific mAbs (NCT02253992). Although CD137-specific and PD1-specific mAbs are well tolerated as single agents, these studies will still need to be followed carefully for synergistic toxicity (for example, hepatitis). The combination of tremelimumab and the CD40-specific superagonist antibody CP-870,893 is also being examined in patients with metastatic melanoma (NCT01103635). In addition, trials are ongoing in patients with advanced-stage solid tumours to evaluate the combination of OX40-specific mAbs with checkpoint inhibitors (MEDI6469) plus tremelimumab or MEDI4737 (NCT02205333), and that of lirilumab with nivolumab (NCT01714739) or ipilimumab (NCT01750580). In these trials, additional clinical caution is recommended, as unexpected severe reactions could occur.

Our current knowledge of the crosstalk among co-inhibitory and co-stimulatory receptors is limited. However, multiple mechanisms for synergy arise when considering that the functions repressed by checkpoints are frequently induced by co-stimulatory molecules on the same target cells. Figuratively, it would be like releasing the brakes while acting on the gas pedal of a car92, 93, 113(Fig. 3). Although combinations of mAbs targeting co-stimulatory molecules (such as CD137-specific and GITR-specific antibodies) with PD1-specific or PDL1-specific antibodies work synergistically in mouse models, it is difficult to predict whether — and, if so, how — this would translate to the human situation. Therefore, these combinations should be evaluated carefully in early clinical trials.

Combining immunomodulation with conventional therapies

Conventional treatment regimens — such as chemotherapy, radiotherapy, targeted therapy and ADCC mediated by tumour-targeting antibodies — may, in causing tumour cell death, allow the release of tumour antigens for presentation and thus prime the immune system92, 93, 114, 115, 116, 117 (Fig. 3). Therefore, it will be important to test the effects of combining immunomodulatory mAbs with these therapies. Reducing tumour burden using conventional therapies may also allow immunotherapy to be more effective. A few key trials have been completed7, 8, 32, 94, 95, 96, 97, 98, 99 (Table 1), and numerous trials are ongoing (see Supplementary information S1, S2, S3 (tables)).

In the pivotal trials, ipilimumab was combined either with a GP100 peptide vaccine or with the chemotherapeutic agent dacarbazine7, 8. Whereas the first combination did not add toxicity or benefit over ipilimumab alone, the dacarbazine and ipilimumab combination conferred a survival benefit over dacarbazine alone. Although mechanistically, dacarbazine, as a DNA-alkylating agent, could lead to tumour cell destruction, release of antigens and sensing by immune cells of tumour-derived DNA, its response rate is too low to expect an important synergistic effect. Therefore, the improved overall survival achieved by the combination compared with dacarbazine alone has been attributed solely to ipilimumab. However, this combination had a high rate of treatment discontinuations, mostly owing to increased levels of liver enzymes. This was considered to be the result of synergistic toxicity, as both drugs have intrinsic hepatotoxicity via different mechanisms: direct liver toxicity by dacarbazine and liver inflammation mediated by ipilimumab. Although the exact mechanism has not been clearly elucidated, this synergistic dose-limiting toxicity is a cautionary note for immuno-oncology combinations. Interestingly, data presented at the 2014 American Society of Clinical Oncology (ASCO) annual meeting showed that a combination of ipilimumab with temozolomide (a drug similar in structure to dacarbazine) did not have such a high rate of dose-limiting liver toxicity, as unlike dacarbazine, temozolomide is not metabolized by the liver118.

Hepatotoxicity resulting from combination therapy was also reported when the BRAF-V600E inhibitor vemurafenib was combined with ipilimumab in a small 10-patient Phase I study119. The rationale for this combination was that MAPK inhibitors not only induce tumour cell death but also lead to the upregulation of MHC class I molecules and melanocyte differentiation antigens, such as melanoma antigen recognized by T cells 1 (MART1), GP100 and tyrosinase120. In addition, BRAF inhibitors and ipilimumab monotherapy had been shown to improve presentation of tumour-specific antigens and increase the infiltration of lymphocytes into the tumour120, 121, also providing a rationale for this combined approach. Preclinical data in mice had shown that inhibiting wild-type BRAF in T cells has no detrimental effect on their viability and function, and even seemed to improve T cell function122. Although the BRAF-V600E inhibitor vemurafenib is known to induce skin toxicity and, more rarely, liver toxicity, grade III elevation of liver enzymes was observed in six of ten patients, and the study was stopped119. As ipilimumab has a half-life of 15 days, administration of vemurafenib within 4 weeks of the last dose of ipilimumab can be considered combination therapy. This may have resulted in an unexpectedly high and severe (grade III) skin rash in a small, sequential therapy study (all of the three treated patients showed skin rashes)123. On biopsy, the rash was consistent with a drug reaction but not autoimmune dermatitis, so the exact pathogenesis remains unresolved. There is currently intensive investigation into PD1 blockade in combination with BRAF inhibitors alone or with BRAF inhibitors and MEK inhibitors (see Supplementary information S1 (table)). In biopsy samples taken shortly after the initiation of BRAF-V600E inhibition in patients with melanoma, there were clear signs of an influx of TILs122, providing a rationale for combining BRAF inhibitors with immunotherapies such as PD1 blockade.

Ipilimumab has also been combined with the vascular endothelial growth factor (VEGF) inhibitor bevacizumab, which inhibits tumour angiogenesis. In addition to its pro-angiogenic function, VEGF has immune-modulating properties, which include decreasing the influx of lymphocytes and DCs into the tumour, while increasing the intratumoural frequencies of TReg cells and myeloid-derived suppressor cells (MDSCs). Hodi et al.17 recently reported a trial combining ipilimumab and bevacizumab in patients with metastatic melanoma. In total, 46 patients were treated with this combination, and the efficacy was remarkably good, resulting in a median overall survival of more than 2 years. High-grade toxicity was more common than expected for either drug alone, but it was manageable and included hypophysitis, temporal arteritis, dermatitis and hepatitis. Interestingly, the combination led to an accumulation of CD8+ T cells and DCs in the tumour microenvironment — suggesting synergism of immunotherapeutic effector mechanisms — and warrants further investigation of this combination.

In radiotherapy, the abscopal effect describes a rare event of regression of metastatic lesions distant and outside of the irradiation field. A patient with metastatic melanoma who was treated with ipilimumab and then showed tumour progression at several sites was given radiotherapy to target one painful metastasis. Thereafter, the patient responded not only at the irradiated site but also at other lesions124. The patient's tumour expressed the cancer–testis antigen NYESO1, and during ipilimumab treatment, the levels of NYESO1-specific antibodies in their serum slowly increased. After radiotherapy, antibody titres against various antigens rose rapidly concurrently with tumour regression, suggesting that an immunological response had been triggered by the radiotherapy while on ipilimumab maintenance treatment. This observation has recently been confirmed by others125. Mechanistically, the combination of stereotactic radiotherapy followed by CTLA4 blockade was dependent on CD8+ TILs; resistance to the combination was predicted by a low CD8+ T cell/TReg cell ratio, expression of PDL1 by tumour cells and the presence of CD8+ T cells with an exhausted phenotype126. Part of the resistance could be overcome by adding PDL1-specific antibodies to the combination of stereotactic radiotherapy and CTLA4 inhibition. In support of this, a study of a small number of patients receiving ipilimumab and radiotherapy showed evidence of benefit when some tumour lesions were irradiated with hypofractionated doses126.

Managing combination-associated toxicity

Checkpoint-targeted immunotherapies can induce a new class of adverse effects as a result of supraphysiological immune activation that may overwhelm key organ tolerance mechanisms127,128, 129. These immune-mediated adverse events mimic autoimmune diseases (such as dermatitis, inflammatory colitis, thyroiditis, hypophysitis and autoimmune hepatitis). However, they lack the chronicity that is often associated with true autoimmunity and, at least for gastrointestinal adverse events, seem to have different pathophysiology from classic inflammatory bowel disease130. Clinically apparent dermal and mucosal inflammation might also result from overactive immune responses to antigens of the commensal flora. Each immunotherapeutic class of drugs is associated with adverse events10 that can be clinically managed but that in certain combinations might surpass the threshold of tolerability.

Studies of combined PD1 and CTLA4 blockade in melanoma and other tumour types suggest that, with sufficient clinical experience and appropriate management algorithms, immune checkpoint inhibitors can be safely given to patients11, 110, 111. Little is currently known about the long-term effects of combination therapy, and whether a different range of immune-mediated toxic effects will manifest with chronic exposure. In the case of CTLA4 blockade, unexpected ectopic expression of CTLA4 has been reported in the adenohypophysis. This expression may explain the hypophysitis cases described during CTLA4-specific mAb treatment that occurred, at least in part, as a result of complement fixation by the mAb131.

Immunotherapy combinations can also be used to reduce the adverse events caused by monotherapy. Based on preclinical mouse models, it was hypothesized that combining granulocyte–macrophage colony-stimulating factor (GM-CSF) with ipilimumab may result in synergistic antitumour activity through increased inflammation of the tumour. In a recent Phase II clinical trial that assessed the combination of ipilimumab and subcutaneous GM-CSF, the incidence and severity of immune-mediated adverse events associated with ipilimumab were unexpectedly mitigated by this combination. Results from this randomized study showed improved 1-year survival and overall survival in patients with metastatic melanoma when compared with ipilimumab monotherapy95. Interestingly, gastrointestinal toxic effects in particular were observed to be significantly less frequent and less severe in the combined treatment group compared with in patients treated with ipilimumab alone. As GM-CSF is important for the induction of immune regulation in the gut, the recombinant GM-CSF (sargramostim) may have functioned to protect the gut mucosa from ipilimumab-induced mucositis in this trial.

Biomarkers for combination immunotherapies

Challenges to identifying biomarkers

Predicting optimal immunotherapy combinations accurately and confirming their clinical activity in cancers will require the identification and validation of reliable surrogate biomarkers. Several approaches and data sources can be used for identifying and prioritizing immunotherapy combinations, but there are important considerations for each of them100, 101 (Fig. 5a). Animal models have advantages in terms of their tractability and can be useful for identifying combination therapies. For example, syngeneic mouse tumour models showed synergy of CTLA4-specific and PD1-specific antibodies132 (M. Selby, J. Engelhardt, L.-S. Lu, M. Quigley, C. Wang, B. Chen and A.J.K., unpublished observations) and, in a transplanted murine melanoma model, CTLA4-specific antibodies induced an increase in the number of PD1+ and PDL1+ TILs103, 132, providing a rationale for combining ipilimumab and nivolumab in patients11. However, these models are unlikely to recapitulate the complex interactions between the human immune system and a heterogeneous tumour that has undergone immune editing133.

The general consensus holds that many immunotherapy biomarkers are in the tumour microenvironment134, 135, 136 (Fig. 6a) and thus may require invasive repeated biopsies45, 137,138, 139, 140. Relying on immune parameters from peripheral blood is attractive owing to the ease of sampling, but it may not reflect the local tumour microenvironment where the immune contexture is crucial. In addition, potential biases may be introduced by the site of tumour sample collection. For example, samples of superficial cutaneous and lymph node metastases may be the easiest to collect; however, their biology may differ from the primary tumour or from metastases at other sites141. In melanoma tumour staging, the location of metastases is itself prognostic: visceral metastases are associated with a worse prognosis than non-visceral metastases142. In addition, PDL1 expression varies by melanoma location143.

Figure 6: Biomarker discovery for combination immunotherapy and proposed new concepts for clinical management with immunotherapy based on biomarkers.

Evaluating post-treatment tumour samples is also crucial for identifying pharmacodynamic changes that are induced by immune-checkpoint inhibition. However, the timing of the biopsy may influence the results. Sampling at a fixed time point early after treatment may not allow enough time for immune activation, and bias may be introduced by the sample location, although the data could offer insight into the early steps in immune activation. An alternative approach would be to identify and sample lesions that are regressing or progressing regardless of time since therapy. Such an approach may capture the ultimate mechanisms of antitumour inflammation and/or tumour resistance.

Assessment of tumour samples by flow cytometry and functional analyses on cell suspensions are very difficult to carry out, primarily owing to the limitations of sample volume. However, these analyses may prove to be most informative, as they can provide functional data on specific immune cell types, rather than on the tumour as a whole), and they allow more accurate quantification than is generally achievable by using immunohistochemistry (IHC) and gene expression profiling. Technological developments using multicolour tissue immunofluorescence should be incorporated to maximize the information obtained from limited biopsy specimens. Recent examples include: a functional evaluation of the TIL response to neoantigens in a patient with advanced-stage melanoma who had a clinical response to ipilimumab144; and the observation that ipilimumab treatment induced a significant number of newly detected T cell responses but only infrequently boosted pre-existing responses107. More recently, similar correlative data have been found that indicate an association between the number of non-synonymous mutations giving rise to self-human leukocyte antigen (HLA)-presented tumour neoantigens and the response to pembrolizumab in patients with NSCLC145 (Box 2). In one of the responders to PD1 blockade, CD8+ T cells against a neoantigen expressed by the tumour became detectable after initiation of treatment, and the frequency measured in peripheral blood was highly correlated with the developing objective antitumour response, strongly suggesting a role for these T cells in the clinical benefit observed.

As detailed in Box 2, the presence and abundance of non-synonymous somatic mutations that give rise to MHC-presented antigens are crucial to the effect of immunotherapies. This is the interpretation of the positive results from a recent Phase II clinical trial that was designed to evaluate the PD1-specific mAb pembrolizumab in a relatively small, but very informative, series of patients with colorectal and those with non-colorectal cancer whose tumours harboured mismatch-repair deficiencies146. In such mutation-prone cases, PD1-specific therapy achieved a much more superior overall response rate and higher rates of progression-free survival than in cases without mismatch-repair deficiencies.

Therapeutic biomarkers in the tumour microenvironment

For many cancer types, the presence of immune infiltrates — especially T cells — is associated with improved survival, as it illustrates the presence of an ongoing immune response147, 148, 149, 150, 151. An inflammatory tumour microenvironment, especially the presence of CD8+ T cells at the invasive tumour margins, may predict a response to immunotherapy with PD1-specific or PDL1-specific antibodies45, 138. Other immune cell types recruited to the tumour can suppress an antitumour immune response. Tumours are rendered resistant to attack owing to the expression of PDL1 and the production of transforming growth factor-β (TGFβ), indoleamine 2,3-dioxygenase 1 (IDO1) and other immunosuppressive compounds and molecules by tumour-associated macrophages (TAMs), immature tumour-associated DCs (TADCs), TReg cells, interleukin-10-producing regulatory B cells, MDSCs and tumour cells themselves152, 153, 154. The presence of some of these immune-inhibitory molecules — in particular, high expression of PDL1 by tumour immune infiltrates (as well as high expression of CTLA4 and fractalkine (also known as CX3CL1)) — seem to predict responses to PD1- or PDL1-specific treatment in several tumour types, including bladder cancer, NSCLC, melanoma and renal cell carcinoma; it also reflects interferon-γ (IFNγ) expression by tumour-specific T cells, hence representing an ongoing immune response45, 155. IHC and genetic profiling of the tumour microenvironment may be used to categorize cancers according to their immunosuppressive mechanisms and thus rationally treat them.

Tumour samples taken sequentially, before and after treatment, have been analysed to elucidate the mechanisms of action of CTLA4 and PD1–PDL1 inhibition107, and such approaches of single immunotherapy may help to guide the selection of combination immunotherapies45, 107, 138, 144,145, 156, 157, 158, 159, 160, 161 (Table 2). In these studies, the collection of baseline tumours was generally proximal to administration of the CTLA4-, PD1- or PDL1-specific therapy. A randomized Phase II study of 82 patients with melanoma treated with 3 mg or 10 mg per kg ipilimumab showed that patients presenting objective responses or stable disease for ≥6 months tended to have higher expression of forkhead box P3 (FOXP3) and IDO1 at baseline, as determined by IHC, than patients without clinical activity156, 157. Gene expression profiling of the tumour samples showed that patients with clinical activity had higher baseline expression of immune-related genes than those without clinical activity157. The association between IDO1 expression and benefit from ipilimumab may be validated by two ongoing clinical trials that are evaluating IDO1 inhibitors in combination with ipilimumab in patients with advanced-stage melanoma (NCT01604889 and NCT02073123).

Table 2: Immunotherapy biomarkers in the tumour microenvironment

Clinical studies have also shown that CTLA4 inhibition leads to increased numbers of TILs, as assessed by IHC156, 160, 162 and gene expression profiling157. These TILs include memory T cells162 and probably result in an increase in the expression of the genes encoding IFNγ and other TH1 cell-associated proteins157. In addition, these studies also showed possible associations with clinical activity relative to baseline biopsies in TILs156, CD8+ T cell/FOXP3+ T cell ratios160 and the expression of immune-related genes157. However, these studies have generally only identified associations and do not constitute identification of a definitive biomarker. Furthermore, the intratumoural spatial distribution of inflammation may be an important feature to characterize138, 139.

The combination of ipilimumab and nivolumab was supported by preclinical studies with surrogate antibodies103, 132 and by the assessment of human tumours following treatment with ipilimumab or PD1-specific therapy, which showed an increase in the number of TILs and in the expression of IFNγ-inducible genes156, 157. The expression of CTLA4 in pretreated tumours also correlated with more frequent clinical responses to PDL1-specific treatment45. In addition, analysis of metastatic melanoma samples showed that PDL1+ tumour cells colocalize with both TILs and IFNγ expression153, and that pretreated tumours with elevated baseline expression of IFNγ-inducible genes — including IDO1 — seem to respond to PDL1 blockade45; this further supported clinical trials with combination ipilimumab and nivolumab therapy153.

Although biomarkers can be used to develop algorithms that predict the probability of responses to immunotherapies (for example, in PDL1 and PD1 blockade in melanoma138), the value of predictive markers may change markedly with combination therapy. For instance, in melanoma biopsy samples taken from patients before treatment PDL1 expression lost its partial predictive value at least in estimating overall response rate and progression-free survival in patients undergoing concomitant CTLA4 and PD1 blockade11, 98, 99. Therefore, using biomarkers to guide the clinical management of combinations134, 135, 136 (Fig. 6b) is uncharted territory that remains to be developed, but it will probably represent a dramatic shift in our clinical strategies. The value of PDL1 expression in tumour tissue determined by IHC as a predictor of response is also challenged by a recent report of the extraordinary clinical benefit with nivolumab as second-line monotherapy for patients with squamous NSCLC163. In this study, the expression of PDL1 was neither prognostic nor predictive for benefit with nivolumab. In addition, for PDL1 expression in the microenvironment to become of value to clinical decision making, effort is needed to harmonize and validate the different IHC assays developed by academia and by pharmaceutical companies.

Further studies of tumour samples at baseline and after treatment, including lesions that progress with other immunotherapeutic agents, may continue to help to prioritize optimal combinations, providing that relevant targets are expressed. A crucial unmet need is the development of non-invasive imaging techniques designed to assess immune infiltrates.


Two main types of combination regimens are under investigation and will provoke much activity in the near future: combinations of immunotherapies with standards of care (see Supplementary information S2 (table)) and, more excitingly, combinations of immunotherapies among themselves, chiefly involving PD1–PDL1 blockade as a partner (see Supplementary information S1, S2, S3 (tables)). The art of finding markedly synergistic effects at present is empirical rather than rational, mainly because the complexity of the mechanisms of action involved means that the overall effect is difficult to predict and model in experimental systems. Decisions to move new combinations into the clinic are to be informed by preclinical data in animal models, mechanistic evidence for pharmacodynamic interactions and selection of patients based on biomarkers primarily found in malignant tissue biopsies. However, clinicians should acknowledge that the ability to predict which combination is the best for a given specific malignant indication, or for a given patient, is currently rather limited.

To make the most of this landscape of opportunities135, 136, scientists and clinicians need to work together to actively discover and develop new agents as partners for combination and take advantage of biotechnological advances to produce improved, next-generation immuno-oncology agents. Consideration needs to be given to delivery routes of immunotherapies to maximize their bioavailability in tumours and tumour-draining lymph nodes164, 165. Agents with low efficacy as monotherapy, but with multiplicative potential for synergistic effects, should also be tested. In addition, it will be important to consider the optimal types of trial design for combination therapies94, 99 (Fig. 5b) (for example, it may be necessary to extend signal-seeking trials to demonstrate efficacy)135, 166. Revisions of regulatory rules may be needed to evaluate and demonstrate the efficacy of combinations in clinical trials for approval; for example, more meaningful end points should be considered, such as overall survival at a given milestone following treatment (that is, overall survival at 1 year or 2 years post-treatment).

On the basis of currently available efficacy data, we predict that most immunotherapy combinations will be built on PD1–PDL1 blockade (Fig. 4). Therefore, thorough study of the limiting factors for this molecular strategy will help in the quest for selection of optimal combination partners. These may include strategies to prime T cell responses, including vaccines103 and adoptive T cell therapy directed to neoantigens presented by tumour cells167, 168; inducers of immunogenic cell death in strategies of in situ vaccination92, 169, 170; simultaneous targeting of checkpoint inhibitors responsible for T cell anergy and/or exhaustion11, 113, 171, 172; and simultaneously providing artificial co-stimulatory132, 173, 174 and/or local pro-inflammatory agents175, 176. Ideally, these therapies should be selectively targeted to the tumour microenvironment and tumour-draining lymphoid tissue164, 165.

Regardless of their complexity and the issues associated with designing optimal combinations, immunotherapy combinations may be perceived as a leading way to increase therapeutic success across a whole range of tumour types.


The authors take full responsibility for the content of this publication and confirm that it reflects their viewpoint and medical expertise. The authors wish to acknowledge R. Turner and K. McGlynn of StemScientific, an Ashfield Company, part of UDG Healthcare plc, funded by Bristol-Myers Squibb, for coordinating the writing process and providing editorial support.

Author information


Centro de Investigación Médica Aplicada (CIMA) and Clínica Universitaria, Avenida Pío XII, 55 E-31008, Universidad de Navarra, Pamplona, Spain.
Ignacio Melero, M. Angela Aznar & José Luis Pérez Gracia

Bristol-Myers Squibb, 3551 Lawrenceville Princeton, New Jersey 08648, USA.
David M. Berman

Bristol-Myers Squibb Biologics Discovery California, 700 Bay Road, Redwood City, California 94063, USA.
Alan J. Korman

The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands.
John Haanen

Competing interests statement

D.M.B. and A.J.K. are full-time employees of Bristol-Myers Squibb. I.M, M.A.A, J.-L.P.G. and J.H. declare no competing interests.

Corresponding author

Correspondence to: Ignacio Melero

Author details

Ignacio Melero earned an M.D. degree from the University of Navarra School of Medicine, Pamplona, Spain, in 1988 and was trained as a resident in clinical immunology at Hospital de la Princesa (Madrid, Spain). He also attained a Ph.D. degree working with Miguel Lopez-Botet, pioneering the characterization of natural killer cell inhibitory receptors and CD94. In 1994, he moved to Seattle (Washington, USA), where he worked on tumour immunology and immunotherapy. His studies of that time on CD137-mediated stimulation of curative antitumour immune responses have received much attention by the cancer immunotherapy community. In 1998, he returned to Navarra University, where he works at Clínica Universitaria and at the Centro de Investigación Médica Aplicada (CIMA). His current areas of translational research are focused on cell-, gene- and monoclonal antibody-mediated immunotherapies for cancer.

David M. Berman received his M.D. and Ph.D. from the University of Texas Southwestern Medical School (Dallas, Texas, USA). He then completed a residency at the National Cancer Institute (Maryland, USA) and a fellowship at the Johns Hopkins Hospital (Maryland, USA) in anatomical pathology. He joined Bristol-Myers Squibb (New Jersey, USA) in 2005, where he worked on the early-phase clinical and translational studies of ipilimumab. He has continued to work in immuno-oncology over the past 10 years, including as global clinical lead for elotuzumab and ipilimumab. He is currently Head of the Immuno-Oncology Exploratory Development Team and is responsible for developing the early-stage immuno-oncology portfolio of drugs at Bristol-Myers Squibb.

M. Angela Aznar earned her degrees in biology and biochemistry in 2005 and 2006, respectively. In 2008, she obtained a doctorate grant from the Spanish Ministry of Education and Science and earned her Ph.D. in biochemistry at the Centro de Investigación Médica Aplicada (CIMA), Pamplona, Spain, in March 2011. Her studies dealt with the molecular characterization of mucosa-associated lymphoid tissue and with diffuse large B cell lymphoma. In 2012, she joined the Pharmaceutical Technology Department of Navarra University, Pamplona, Spain, where she worked on nano-sized drug delivery systems as anticancer therapies for breast cancer and leukaemia. She is currently a research scientist in Ignacio Melero's team at CIMA, where she is working on translational research in the areas of tumour immunology and immunotherapy.

Alan J. Korman received his Ph.D. degree from Harvard University (Cambridge, Massachusetts, USA) in 1984 and was a Whitehead Fellow at the Whitehead Institute, Massachusetts Institute of Technology (Cambridge, Massachusetts, USA) from 1984 to 1989. He was also a Chargé de Recherche at the Institut Pasteur (Paris, France) from 1990 to 1993. He has worked in the biotechnology and pharmaceutical industry since 1993 and is currently Vice President of Immuno-Oncology at Bristol-Myers Squibb (California, USA). He initiated the preclinical development of ipilimumab and nivolumab.

José-Luis Pérez Gracia received his M.D. from the Free University of Madrid (Spain) in 1994. He performed his residency in medical oncology in Hospital 12 de Octubre in Madrid from 1995 to 1998 and obtained his Ph.D. from University Complutense (Madrid) in 2001. From 1999 to 2003 he worked in the Clinical Research Department of Eli Lilly & Company (Madrid, Spain). In 2003, he moved to the University Clinic of Navarra (Pamplona, Spain) where he became Associate Professor of Medicine and currently leads the Urologic Cancer and Clinical Research Units. He has participated in many academic and industry-sponsored immunotherapy clinical trials and many translational research projects, especially in the field of biomarker development and cancer immunotherapy.

John Haanen studied at Leiden University, Netherlands, and received his M.D. in 1988. His Ph.D. work was performed at the Leiden University Medical Center (LUMC) and at DNAX Research Institute (Palo Alto, California, USA) and involved the characterization of the human T helper cells that target mycobacterial antigens. Following his internal medicine training at the LUMC, he moved to the Netherlands Cancer Institute in Amsterdam in 1997 for a postdoctoral fellowship, during which time he worked on influenza virus-specific CD8+ T cells with Ton Schumacher, as well as on other projects. He subsequently trained as a medical oncologist and received a permanent position in Amsterdam. His current areas of translational research are the development of therapeutic DNA vaccines, adoptive cell therapy with tumour-infiltrating lymphocytes, and T cell receptor gene-modified T cells.

Supplementary information

PDF files

  1. Supplementary information S1 (table) | Combinations of PD-1/PD-L1 antagonistic antibodies with conventional anticancer therapy (Source: accessed on May 2, 2015) (117 KB)
  2. Supplementary information S2 (table) | Combinations of immunoregulatory antibodies (excluding PD-1/PDL1 antagonistic antibodies, see Suppl. Table 1) with conventional anticancer therapy. (Source: accessed on May 2, 2015). (148 KB)
  3. Supplementary information S3 (table) | Combinations including two or more immunotherapy agents based on PD-1/PD-L1 blockade. (Source: accessed on May 2, 2015). (129 KB)


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