Combination of checkpoint inhibitors with other agents as a strategy to improve anti-cancer effect – a glimpse to the future

ABSTRACT

In the last years, a remarkable progress has been made in the clinical application of novel immunotherapy agents, the so called ‘checkpoint inhibitors,’ that has revolutionized the treatment of many malignant tumors. Their design has been based on the immune-mediated mechanisms of antitumor activity circle, such as antigen release and presentation, activation and trafficking of T-cells into tumors, depletion of immunosuppression, and immunogenic cell death. Various combinations of checkpoint inhibitors are being designed and/or tested, such as double checkpoint blockade, combination with chemotherapy, radiotherapy, molecularly targeted agents, and other immune-directed strategies.

KEYWORDS:

• Immunotherapy
• cancer
• PD-1
• ckeckpoint inhibitors
• immunotherapy combinations

1. Introduction

In the last years, remarkable progress has been made in the clinical application of novel immunotherapy agents, the so called ‘checkpoint inhibitors,’ that has revolutionized the treatment of malignant tumors [1]. Their design has been based on the immune-mediated mechanisms of antitumor activity circle, such as antigen release and presentation, activation and trafficking of T-cells into tumors, depletion of immunosuppression, and immunogenic cell death (ICD) [2]. Various combinations of checkpoint inhibitors are being designed and tested, such as double checkpoint blockade, combination with chemotherapy, radiotherapy, molecularly targeted agents, and other immune-directed strategies [3].

2. The role of checkpoint pathways in cancer evolution

T cells mediated cellular immunity is controlled by a balanced system that functions through stimulatory and inhibitory proteins [2]. The inhibitory receptors, also known immune checkpoints, regulate T cell activation and effector functions to sustain self-tolerance and minimize bystander tissue damage. When T cell receptor (TCR) distinguishes antigens in the presence of major histocompatibility complex (MHC), the immune checkpoint molecules modulate signaling of co-stimulatory factors to amplify the signal, whereas co-inhibitory molecules suppress it. It has been shown that the expression of immune-inhibitory checkpoints function as potent mediators for sustaining a balance (Figure 1). These molecules are expressed on activated T cells, but when they adhere to ligands either on antigen presenting cells (APCs) (CTLA-4 binding to CD80/CD86) or tumor cells (PD-1 binding to PD-L1), they tend to suppress the anti-tumor response [4]. Efforts to use monoclonal antibodies (mAbs) to target these immune-inhibitory interactions have led to a new era of cancer immunotherapy (Table 1). PD-1 associated immune-resistance depends on the accessibility of PD-L1 ligand in the tumor. The PD-L1 expression is regulated either by upregulation of PI3K-AKT cascade or secretion of IFN-γ [5]. Two types of immune resistance have been observed, innate and adaptive immune resistance [6].

Table 1. Currently approved checkpoint inhibitors.

Mode of action	Kind of molecule	Malignant tumor
CTLA-4 targeting
Ipilimumab	Fully humanized mAb	Malignant melanoma Kidney cancer
PD-1 targeting
Nivolumab	Fully human IgG4 mAb	Malignant melanoma Kidney cancer Non-small cell lung cancer Head and neck squamous cell cancer Urothelial cancer Hodgkin’s lymphoma Colorectal cancer with MSI-H
Pembrolizumab	Humanized IgG4 mAb	Malignant melanoma Non-small cell lung cancer Urothelial cancer Tumors with MSI-H Head and neck squamous cell cancer
PD-L1 targeting
Durvalumab	Human IgG1 mAb	Urothelial cancer
Atezolizumab	Fully humanized IgG1 mAb	Urothelial cancer Non-small cell lung cancer
Avelumab	Human IgG1 mAb	Merkel cell carcinoma Urothelial cancer

Figure 1. Checkpoints implication in malignant immune-surveillance and potential ways of combining checkpoint inhibitors with other strategies.

When a T cell recognizes an antigen presented by the MHC on the target cell, inflammatory cytokines are produced. These cytokines result in PD-L1 tissue expression and activation of the PD-1 protein on T cell leading to immune tolerance, a phenomenon where the immune system lose the control to mount an inflammatory response, even in the presence of actionable antigens [7]. It has been found that PD-1 is expressed on a variety of immune cells, such as monocytes, T cells, B cells, dendritic cells (DCs), and tumor-infiltrating lymphocytes (TILs). However, PD-L1 is mainly expressed in tumor cells and APCs [8]. The function of tumor cells that overexpress PD-L1 is to protect themselves from cytotoxic T cell (CD8+) mediated cell killing. Due to exhaustion of CD8 + T cells, tumor cells become very aggressive and secrete several pro-inflammatory cytokines, such as tumor necrosis factor alpha (TNF-α), interleukin-2 (IL-2), and interferon gamma (IFN-γ) [9]. PD-1 has two binding partners, namely PD-L1 (B7-H1) and PD-L2 (B7-DC). The binding affinity of PD-1 with PDL-1 is three times greater that the affinity between PD-1 for PD-L2. PD-L2 has restricted expression on macrophages, DCs and mast cells and its’ role in cancer immunosuppression is still obscure [10].

Another subtype of T cells, regulatory T cells (Tregs, CD4+ FOXP3+) and MDSCs (myeloid-derived suppressor cells), create a highly immunosuppressive tumor environment through maintaining the expression of PD-1 on their surface [11]. It has been found that in the presence of CD3 and TGF-β, the PD-1 receptor of Tregs increases the de novo transformation of naïve CD4 + T cells to Tregs, thus attenuating immune responses. This conversion increases Tregs expression and immune suppressive function of CD4 + T-cell through inhibition of PI3K/AKT signaling cascade [12].

Another important checkpoint molecule, CTLA-4, is broadly engaged in tumor immune evasion through the down-regulation of CD4 + T effector cells and the enhancement of Tregs activity [13]. CTLA-4 binds to the B7-1/2 protein of APC and determines whether T cells will undergo activation or suppression. It is believed that CTLA-4 binding to B7 yields T-cell inhibitory signals, which also depends on stimulating TCR and MHC antigen binding.

3. The current role of checkpoint inhibitors in cancer therapeutics

PD-1 and PD-L1 targeting is an effective way to maintain the function of effector T-cells. Currently, it is not clear whether PD-1 or PD-L1 inhibitors are more effective, since it depends on patients’ characteristics, type of tumor, genetic defects (e.g. EGFR mutations, ALK translocations) and site of metastases [1]. As tumor is heterogeneous, the expression of PD-L1 is not uniform, thus PD-L1 immunohistochemistry staining varies. Therefore, PD-L1 expression and response of PD-L1 targeting agents remain a debatable correlation. The same is the issue with PD-1 expression and the efficacy of PD-1 targeting agents.

Single checkpoint blockade has demonstrated good anti-tumor activity in many malignant tumors (Table 1). However, the survival gain remains low. Therefore, various combinatorial strategies have emerged as a promising strategy to improve clinical outcomes (Figure 1s).

4. Combination of checkpoint inhibitors with other immunotherapy strategies

Most of the benefits of anti-CTLA-4 blockade seem to be mediated by Tregs depletion. Therefore, the combined inhibition of PD-1/PD-L1 and CTLA-4 is being evaluated as a more potent anti-tumor treatment strategy with impressive, so far, clinical results [14,15]. Antibodies against other checkpoint receptors, including LAG-3, TIM-3 have also entered clinical testing alone or in combination with anti-PD-1/PDL-1 agents [6]. Additionally, agonistic antibodies can enhance T-cell effector function, proliferation and survival as well as boost CD8 + T-cell differentiation and overcome Tregs suppression (e.g. anti-OX40, anti-CD27, anti-CD137) [6]. These agents have shown pre-clinical activity and have entered clinical testing alone or in combination with checkpoint inhibitors.

Tumor cells compete with T cells for essential nutrients and they can up-regulate IDO-1, an important immunomodulatory enzyme in tryptophan catabolism in order to promote Tregs activity and suppress T-cell function [16]. IDO-1 inhibitors are currently tested in combination with checkpoint inhibitors since it has been shown in pre-clinical models that they can enhance their action.

Historically, the major drawback of vaccines tested in cancer treatment has been the phenomenon known as ‘T cell exhaustion.’ It manifests a progressive up-regulation of inhibitory checkpoint molecules and progressive loss of T-cell effector functions. Additionally, influx of interferon-secreting T-cells into the tumor leads to the up-regulation of PD-L1 on tumor cells, an adaptive mechanism through which tumor cells can evade immune surveillance [9]. The combination of cancer vaccines with checkpoint inhibitors constitutes a rational approach, although the appropriate sequence of this combinatorial approach remains an open question.

Oncololytic viruses (OVs) promote anti-tumor response through direct tumor cell killing as well as the induction of innate and adaptive anti-tumor immunity. Theoretically, OVs can enhance checkpoint blockade as it has been shown in animal models, but this remains to be proved in clinical trials that are ongoing [9].

The most direct way of triggering T-cell presence in the tumor is by adoptive cell therapy (ACT). Among the various ACT approaches, synthetic chimeric antigen receptors (CARs) have gained most scientific interest. Unlike TCR that is HLA-restricted, CARs can bind virtually any cell surface-expressed molecule. Therefore, they represent a promising treatment strategy especially for tumors bearing defects in antigen presentation process. After their initial impressive clinical results in hematologic malignancies, they are now entering clinical testing in solid tumors either alone or in combination with checkpoint inhibitors [17].

5. Combination of checkpoint inhibitors with chemotherapy

Several chemotherapeutic drugs promote ICD that is characterized by the secretion of damage-associated molecular patterns (DAMPs), the activation of DCs, and the recruitment and activation of TILs [18]. Furthermore, chemotherapy can induce the expression of T-cell attracting chemokines that have been shown to exert a synergistic effect with checkpoint inhibitors [19]. It remains to be determined if previously or concurrently administered chemotherapy (and which agents) enhance the activity of checkpoint inhibitors.

6. Combination of checkpoint inhibitors with molecularly-targeted agents

Several mAbs that specifically target growth factor receptors preferentially expressed by cancer cells, such as epidermal growth factor receptor (EGFR) protein members, have been approved for use in cancer treatment. Besides inhibiting signal transduction cascades, these mAbs have several immune effector mechanisms, including complement-dependent cytotoxicity and antibody-dependent cellular cytotoxicity (ADCC) [20]. Some mAbs also exert unexpected immune-stimulatory functions. Moreover, other small molecule targeted agents have been shown to mediate therapeutic effects by promoting tumor-targeting immune responses [19,21]. Several anti-angiogenic mAbs and small molecule agents have also shown anti-cancer effect mainly by affecting the receptors engaged by vascular endothelial growth factor A (VEGFA). In early clinical trials, anti-angiogenic agents have been shown synergistic activity in combination with checkpoint inhibitors [22]. Some epigenetic modifiers also mediate anticancer effects by promoting a tumor-targeting immune response [23]. A detailed knowledge of the immunomodulatory effects of molecularly targeted agents may have a profound effect on the design of novel treatment strategies in which such molecules will be combined with checkpoint inhibitors.

7. Combination of checkpoint inhibitors with radiotherapy

Radiotherapy (RT) can induce immunological changes both in the tumor and in its’ microenvironment and to potentially induce systemic responses due to anti-tumor immunity promotion (abskopal effect) with several mechanisms, including enhancement of tumor antigens release, exposure of novel tumor antigens, and increase of pro-inflammatory cytokines activating T cells [24]. It can also induce ICD and the release of DAMPs that promote DC maturation and cross-presentation to T cells. In addition, it has been shown to inhibit Tregs. Through its’ action, especially in the microenvironment, RT might facilitate immunotherapy. Recent pre-clinical data have suggested a synergy between RT and checkpoint inhibitors [25]. This combination is currently under investigation to identify the optimal timing of the combination, the optimal RT dose per fraction, and the effect of the combination regarding the irradiated site.

8. Expert opinion

The clinical use of checkpoint inhibitors to date alone or in combinatorial strategies has shown that only few patients will gain actual survival benefit [1,3]. Therefore, there is a need for the identification of reliable and easily reproducible predictive biomarkers. The immune evasive measures used by cancer cells are broadly separated into two categories defined by cellular and molecular characteristics in the tumor microenvironment; an inflamed phenotype that actively suppresses immune activation and a non-inflamed phenotype that passively escapes immune detection. Among the reasons of checkpoint inhibitors failure are insufficient infiltration of activated CD8 + T cells into the non-inflamed tumors, variable population of CD8 + T cells due to intra-tumor heterogeneity, hypoxia and variability of genetic defects of specific signaling pathways. An immunoscore that is based on the level of tumor immune cell repertoire has emerged as a promising predictive model, irrespectively of other clinical and pathological parameters, although the role of the surrounding micro-environment is still obscure.

Once the immune checkpoints have been blocked, the equilibrium between the autoimmunity and immune tolerance will be affected. The term ‘immune-mediated adverse reactions’ describes the side effects of immunotherapy [1]. In general, toxicities with anti-PD-1/PD-L1 mAbs seem to be less common and severe compared with anti-CTLA-4 mAbs, while combinatorial strategies exacerbate toxicity. The longer treatment with checkpoint inhibitors goes along with more severe immune-related adverse events. Therefore, the optimal dose and schedule of these agents, especially in responding patients, is an important, yet unanswered issue.

The novel mechanism of action of checkpoint inhibitors have produced unusual patterns of clinical responses that resemble tumor flare but are more pronounced and frequent. Some cancer patients treated with these agents and whose disease meet the criteria for disease progression based on traditional response criteria, such as RECIST, have been noticed to have late, deep, and durable responses. These observations led to new recommendations regarding the evaluation of the clinical effect of checkpoint inhibitors, often referred to as irRECIST [26]. However, these new criteria have not yet widely applied in trials and clinical practice leading to concerns about the comparability of results across trials and difficulty in every day clinical decision-making.

Declaration of interest

The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

Additional information

Funding

This paper was not funded.