Combination therapy with immune checkpoint inhibitors (ICIs); a new frontier

During the last two decades, tumor immunotherapy has evolved the clinical management of a diversity of tumors even with undesired prognoses [1, 2]. As one of the most eminent eras in the context of tumor immunotherapy, immune-checkpoint inhibitors (ICIs) have engendered remarkable therapeutic outcomes as a result of their broad bioactivity across numerous histological tumor types along with their durable anti-tumor impacts [3, 4]. Among the checkpoint-blocking strategies, inhibition of the cytotoxic-T-lymphocyte-associated protein 4 (CTLA-4 or CD152) and also blocking the interfaces between programmed cell death 1 (PD-1 or CD279) and programmed cell death ligand 1 (PD-L1 or CD274 or B7 homolog 1) has gained increasing attention [5]. Due to the substantial homology to the costimulatory molecule CD28, CTLA-4 can bind B7 molecules on antigen-presenting cells (APCs) with much higher affinity and also avidity than CD28, averting the activation of T cell responses [6]. The evidence regarding the CTLA-4 activities offered the concept that dampening its activities could enable durable T cell responses [7]. Then, accumulating evidence supported the responding notion, and after than much effort was spent to produce ipilimumab, a monoclonal antibody (mAb) targeting human CTLA-4 [8]. Irrespective of inhibition of the costimulation, CTLA-4 inhibitors can also attenuate regulatory T (Treg) cell recruitment into tumor tissue due to the high expression of CTLA-4 on the surface of Treg [9]. Negative regulation of Tregs population in the tumor microenvironment (TME), in turn, largely improves the infiltration as well as anti-tumor activities of tumor-infiltrating lymphocytes (TILs), in particular, cytotoxic T lymphocytes (CTL) [10]. On the other hand, the PD-1 functions as a critical immune checkpoint were documented upon detecting its central ligand, PD-L1, which is found on multiple cell types such as tumor cells, immune cells, epithelial cells, and endothelial cells [11]. Similar to CTLA-4, the PD-1is expressed on induced T cells and contributes to the down-regulation of signaling complicated in antigen recognition by the T cell receptor (TCR) [12]. PD-L1 expression is in association with exposure to interferon-γ (IFN-γ) for example following anti-tumor T helper type 1 (Th1) cell responses, and could ultimately ease tumor cell’s escape from T cell antitumor immunity [13‐15]. Like anti-CTLA-4 antibody ipilimumab, PD-1/PD-L1 inhibitors, surrounding nivolumab, pembrolizumab, cemiplimab, atezolizumab, avelumab, and durvalumab have gained approval from United States Food and Drug Administration (FDA) during the last decade (Fig. 1) [16, 17]. However, tumor resistance to ICIs [18, 19] and also treatment-associated toxicities [20] impede their clinical utility. Recent reports have shown that objective response rate (ORR) in melanoma patients treated with PD-1 inhibitors is only 33%, and also more than 70% of non-small-cell lung carcinoma (NSCLC) patients exhibit no response to ICIs [21]. It has been evidenced that TME in association with other factors supports chronic inflammation, improves immunomodulation, and concomitantly aids pro-angiogenic intratumoral microenvironment, and thereby entices tumor cells evasion from recognition and succeeding elimination by host immunosurveillance [22, 23]. Accordingly, several studies have exhibited that combination therapy with ICIs plus other therapeutic approaches, such as chemotherapy [24‐26], radiotherapy [27, 28], cancer vaccines [29‐31], anti-angiogenic agents [32‐34], HER-2 targeted therapies [35] and also CXCR4 blockade therapy [36, 37] can efficiently circumvent tumor resistance to ICI therapy.

In the present review, we deliver an overview about the therapeutic merits of ICIs as a pioneering tactic in tumor immunotherapy and also discuss recent reports evaluating the combined use of ICIs with other conventional approaches to overcome tumor resistance to ICI, with a particular concentration on last decade in vivo reports.

Communication between immune checkpoints and their responding ligands abrogates T cell activation and resultant anti-tumor immunity by targeting a myriad of signaling axes, in particular, phosphatidylinositol-3-kinase (PI3K)/Akt pathway [38]. As a result, NF-kB and mTOR activation and also IL-2 and Bcl-xL expression are negatively affected in activated T cells [39]. Such events eventually hinder physiological immune reactions against tumor-associated antigens (TAAs). Notably, immune checkpoints and the related ligands are mainly upregulated in the TME and also on the surface of tumor cells, and so underlies blockade of anti-tumor immune response [40, 41].

As known, CD80 (B7-1) and CD86 (B7-2) co-stimulation by CD28 delivers vital stimulatory signals, which eases T cell proliferation and differentiation throughout the induction phase of immunological response [42]. The CTLA-4 co-inhibitory receptor is largely demonstrated on lately activated T cells and creates interfaces with the same ligands as CD28 but with higher affinity [43, 44]. Interrelation between CTLA-4 and CD80/86 impedes T cell activation by both suppressing the formation of a communication between CD80/CD86 and CD28, and also transmitting suppressive signals [45, 46]. Structurally, CTLA-4 includes a unique YVKM motif at the cytoplasmic domain, which brings about inhibitory signaling upon interaction with the Src homology 2 domain-containing protein tyrosine phosphatase 2 (SHP-2) [7]. CTLA-4 inhibits T-cell responses by cell-intrinsic and extrinsic pathways. Intrinsic events involve the suppression of protein translation and cytokine receptor signaling through the induction of the recruitment of phosphatases and ubiquitin ligases [47]. Besides, cell-extrinsic actions comprise the competition for CD28 in binding to CD80/86, the removing CD80/86, secretion of suppressive indoleamine (2,3)-dioxygenase (IDO), and also targeting Treg activities [47]. Other in vivo reports deliver the proof of the hypothesis that CTLA-4 can adjust T-cell infiltration into allografts as well as tumors [48]. Unsurprisingly, elevated levels of CTLA-4 in association with poor prognosis has been found in NSCLC [49‐51], breast cancer [52, 53], nasopharyngeal carcinoma [54], small cell lung cancer (SCLC) [55], prostate cancer [56], thymoma [57], melanoma [58, 59], colorectal cancer (CRC) [60], glioblastoma [61] and osteosarcoma [62].

Anti-tumor T cells following acquirement of cytokine-producing and cytolytic effector competencies can undergo additional negative regulation by an interaction between PD-1 on such cells with PD-L1 on tumor cells or tumor-associated antigen-presenting cell (APC) in the TME [63, 64]. Interaction between PD-L1expressing tumor cells or APC and PD-1 expressing T cells leads ultimately to eliciting signaling by cytoplasmic tail of PD-1, facilitating T cell exhaustion. The cytoplasmic tail of PD-1 includes two tyrosine-based structural motifs, an immunoreceptor tyrosine-based inhibitory motif (ITIM) (V/L/I/XpYXX/L/V) and an immunoreceptor tyrosine-based switch motif (ITSM) (TXpYXXV/I) [65]. The PD-1 suppressive activities depend on the ITSM phosphotyrosine, which in turn, potentiates the recruiting SHP-2 and suppressing downstream signaling pathways like CTL-4 [65, 66]. Various tumors apply this mechanism by up-regulation of PD-L1 which often relates to unfavorable prognosis. Further, expression of PD-1 on some tumor cells has also recently been elucidated [67]. Indeed, interfaces between PD-L1 on tumor cells with PD-1 on immune cells sustain immune escape and tumor development more chiefly by suppression of cytotoxic T lymphocyte (CTL) effector function [68]. Improved expression of PD-L1 on tumors has been validated to intensely correlate with advanced disease state and unfavorable prognosis in melanoma, breast, gastric, ovarian, liver, kidney, pancreatic, and also bladder cancer [68].

Given that ICIs with the goal of targeting CTLA-4, PD-1, or PD-L1 can dampen immune checkpoints-induced inhibitory impacts on T cells biological processes, making further progress to evolve novel ICIs for broader types of malignancies is urgently justified.

It is now generally documented that tumor cells make close interfaces with the ECM, stromal cells, and also immune cells which typically exist in TME. Such cells in TME support evolving chronic inflammation, enhancing immunomodulation, and simultaneously providing a pro-angiogenic intratumoral microenvironment, and thus ease tumor cells escape from recognition and subsequent removal by host immunosurveillance [120, 121]. For eradication of malignant cells, T cells are required to be efficiently induced by dendritic cells (DCs) in peripheral lymph nodes, home to the malignant tissue, extravasate from malignant tissue blood vessels, and finally infiltrate barricades (such as stromal tissue) to encounter cancer cells [122, 123]. Developing tumors mainly barricade these necessities for T cell immunosurveillance for preventing immune cell-elicited tumor eradication. Given that the efficacy of ICIs treatment is principally inspired by T cells, such competent immune escape may ultimately bring about failures in ICIs therapy. A promotion in PD-L1 in the TME by malignant cells and also APCs is thought to be the most communal approach by which malignant cells bypass immune surveillance [124, 125]. The tryptophan catabolism inside the TME also is contributed to the negative regulation of anti-tumor immune responses. In TME, tryptophan catabolism induced by the IDO, which is largely expressed by myeloid-derived suppressor cells (MDSC) and tumor cells, results in making some immunosuppressive metabolites (e.g., kynurenine) [126]. Bothe kynurenine functions and also exhaustion of the vital amino acid tryptophan impede T cell’s clonal expansion and may entice either T cell anergy or apoptosis [126]. Owing to this fact, the combined effects of IDO inhibitors and ICIs have been speculated as a rational plan to provoke TILs and their functional aptitudes in the TME. This intervention can facilitate removing both IDO-expressing and IDO-nonexpressing poorly immunogenic malignant cells [127]. Likewise, the existence of regulatory T cells (Treg cells), T helper 2 (TH2) cells, and MDSCs in TME is an additional impediment, compromising the efficacy of ICIs therapies by suppressing CTL- and T helper 1 (TH1) cell-mediated tumor immunosurveillance [128, 129]. Exhaustion of such cell types has experimentally been exposed to augment anti-tumor immune responses defeating resistance to ICI [21]. Besides, an intrinsic mechanism such as up-regulation of the tumor-inducing WNT-β-catenin signaling pathways may avert TILs and CD103 + DC infiltration into the TME. As evidenced in melanoma, it appears that β-catenin activation could suppress the expression of chemokine chemokine (C–C motif) ligands 4 (CCL4), which is mainly complicated in immune cell infiltration into TME [130, 131]. Besides, loss of phosphatase and tensin homolog (PTEN) is allied with improved levels of CCL2 and vascular endothelial growth factor (VEGF), reduced infiltration of T cells, and finally resistance to PD-1 inhibitors [132]. Thereby, stimulating DCs migration, maturation, and activation by blockade of immunosuppressive factors, such as VEGF, IL-10, and TGF-β efficiently enables sufficient T-cell priming and cooperation with ICI. As well, cyclooxygenase (COX) expression by tumor cells can hinder tumor cell immunosurveillance as a result of up reregulation of the prostaglandin E2 (PGE2) expression, preparing an inflammatory environment for tumor growth [133, 134]. Moreover, COX-2 overexpression mainly improves Treg trafficking into TME. The metabolic interaction between the transformed cells and immune cells also may give rise to the poor response to treatment with ICI, as evidenced by the study of the tumor and immune cell glucose and glutamine metabolism [135]. In fact, glucose and glutamine metabolism up-regulate the PD-L1 expression in transformed cells by the positive regulation of epidermal growth factor receptor (EGFR)/ extracellular signal-regulated kinase (ERK)/C-Jun pathway [135]. Hence, inhibiting tumor glucose or glutamine metabolism by therapeutic molecules in combination with PD-1/PD-L1 blockade therapies may defeat tumor cell resistance to ICIs. On the other hand, janus kinase (JAK) 1/2 loss-of-function mutations are other tools exploited by tumor cells to trigger primary resistance to PD-1 inhibitors by down-regulation of PD-1 expression [136].

The FDA approved atezolizumab and durvalumab for use in combination with chemotherapy for first-line treatment of patients with advanced SCLC. These approvals were rendering consequences derived from two randomized controlled trials, IMpower133 (atezolizumab) [137] and CASPIAN (durvalumab) [109]. These trials revealed increases in OS with anti-PD-L1 antibodies when used in combination with platinum-based chemotherapy as compared with chemotherapy alone [138]. Atezolizumab has also been approved as a first-line NSCLC irrespective of PD-L1 expression in combination with chemotherapy and bevacizumab [139].

As shown in clinical trials (Tables 4 and 5), addition of ICIs to other therapeutic means has been shown encouraging outcomes to treat even metastatic tumors with unfavorable prognosis. However, the intervention-associated irAEs can hurdle their application in the clinic. Skin and colon are the most common organs, while the normal activity of lungs, kidneys, liver, and also heart mainly impaired by ICIs alone or in combination therapies [241]. Though, corticosteroids are usually exploited to ameliorate moderate and severe irAEs, additional immunosuppressive drugs may sometimes be prerequisite [242, 243]. Also, much efforts have recently been spent to determine predictive biomarkers for ICIs response [244]. Meanwhile, PD-L1 expression, microsatellite instability (MSI), high tumor mutational burden (TMB) along with CD8 infiltrates are noted as foremost predictive markers for ICIs response [245‐247]. Taken together, we propose that fulfilling of large-scale trials with further attention to the predictive biomarkers can durably arouse more preferred outcomes with manageable irAEs.