Immune Checkpoint Inhibitors Programmed Cell Death-1/Programmed Cell Death-Ligand1 (PD-1/PD-L1) for Cancer Therapy

Abstract

Monoclonal antibodies that inhibit “immune checkpoint” through programmed cell death-1 and its ligand (PD-1/PD-L1) blockage have proven remarkable therapeutic action toward a range of cancer types. Hence, immunotherapy, binding the immune system to act against malignant tumors, has generated encouraging outcomes in clinical practice. Nevertheless, the robust advantage is not observed in a large number of patients. Recognizing patients that will probably respond and using therapies covering a larger number of patients necessitate an enhanced understanding of the biological action of PD-1 and cytotoxic T lymphocyte antigen (CTLA) at the cell level and reviewing the performed clinical studies and their outcomes to recognize the accumulating proof of its clinical significance. In this chapter, we will discuss and review the clinical and preclinical data regarding Immune Checkpoint Inhibitors PD-1/PD-L1 to recognize the advances and challenges of their implication in clinical practice.

1. Introduction

Twenty-two years ago, Freeman et al. were the first to introduce programmed cell death-1 and its ligand (PD-1/PD-L1) as an immune checkpoint that later on was the immune inhibitor target in cancer therapy. Generally, checkpoints play a role as a brake to slow down the immune function, and it was proposed that inhibition of these checkpoints may stimulate T cells and eradicate malignant cells more effectively [1, 2].

In immune regulation, CD4+ regulatory T cell’s role is well recognized; they suppress autoimmunity and allergic reactions, on the other side, they enable cancer growth by inhibiting immunity against neoplasms [3, 4].

Immune evasion by cancer cells is mediated through acquiring mechanisms of resistance and escape including creating an environment of immunosuppression using immunosuppressive components such as molecules, cytokines of immune suppression type, and cells including CD4+ regulatory T cells to constrain active immunity against the tumor, thus endorsing tumor growth [4]. There is a suggestion of more malleability of the CD4+ regulatory T cell compartment expression with various aspects such as suppression function deactivation, inflammatory cytokines expression, and transcription reprogramming. Even though it is uncertain which element is responsible for CD4+ regulatory T cell malleability, there is probability that precise microenvironments have the inscription on CD4+ regulatory T cell destiny [5].

Numerous checkpoints of immune reaction have been established to bound the hyper-activation of immune cells involved in self-tolerance. One of the significant checkpoints is PD-1 and its ligands, PD-L1 and PD-L2 expressed mainly on the surface and in the body of lymphocytes, in antigen-presenting cells (APCs), and cancer cells and regulate responses toward antigens. Managing PD-1/PD-L actions facilitates the regulation of numerous immune-related disorders including infection, autoimmune disorders, and malignancies [6]. PD-L1 expression is related to microenvironment of T cells, cytokines produced by T cells of helper type, chemical mediators, interferons, and precise characters of gene expression. Continuous stimulation with an elevated level of PD-1 is frequently established in cancer inflammatory infiltrates mainly lymphocytes, as an expression of PD-L1 is utilized by neoplastic cells to escape immune damage. Blocking of these immune checkpoints is exploited to release the prospective of antagonistic therapy against cancer immune response as cancer treatment strategy [7].

Although there is a significant benefit in the outcome of patients attained with PD-L1/PD-1 blocking therapies, resistances are also commonly observed [8].

2. PD-1/PD-L1 immune checkpoint

Programmed cell death-1(PD-1/CD279) has two identified ligands, PD-L1 and 2 (CD274 and CD273 respectively), each with distinct expression patterns and regulations, of which PD-L1 is expressed in numerous cancers [9]. PD-1 is a transmembrane glycoprotein type 1 of the immunoglobulin superfamily, out of its total 288 amino acids, a 20% amino acid shows distinctiveness to cytotoxic T lymphocyte antigen 4 (CTLA4) and it is encoded by the PDCD1 gene on chromosome 2. PD-1 is formed of an extracellular domain of IgV-like and a transmembrane section. Its tyrosine-based switch motif (ITSM) and inhibitory motif tyrosine base are forming the tail which is located intracellularly. It was considered a CD28 receptor family member of T cells accessory molecules. Although PD-1 has similarities to the CD28 family, it shows distinctive properties distinguishing it from members of such family [10, 11].

PD-1 expression on effector T cells is mediated due to stimulation of the T cell receptor (TCR) and it plays a role as a receptor of immune inhibition. It binds the PD-L1 B7 homologs (B7-H1) and PD-L2 (B7-DC), existing mainly on APCs, and can be prompted in other tissues by cytokines of inflammation [1, 6]. PD-1 has a tendency concerning moderate local activation of T cells in tissues of the periphery. PD-1 may have delayed actions in the T cell activation and decay. Generally, PD-1/PD-L plays a significant role in sustaining T cell self-tolerance with the prevention of autoimmunity, and it reduces some anti-apoptotic molecules expression such as B-cell lymphoma-extra-large (BCL-XL) in addition to pro-inflammatory cytokines secretion [12, 13]. The inhibitory function of PD-1/PD-L1 is mediated primarily on effector T cells but also it acts on regulatory T cells by affecting phosphatidyl-inositol 3 kinase (PI3K) to control T cell autoimmunity and tolerance. In general, tolerance is the failure of T cells that may be manifested as ignorance (failure of activation), or “anergic” status (responding cells are persistent in a refractory status), or deletion (apoptosis of T cell) [3, 14].

PDCDL1 gene on chromosome 9 is responsible for PD-L1 coding in humans, PD-L1 a transmembrane protein type 1 was documented as a member of the family B7 protein. The length of PD-L1 is 290 amino acids of 40 kDa protein. PD-L1 consists of extracellular domains (IgV-like & IgC-like), a transmembrane domain (hydrophobic), and a 30 amino acids cytoplasmic tail. The PD-L1 constitutive expression can be detected at low levels, on inactive lymphocytes, APCs, and in syncytiotrophoblasts, corneal, endothelial, keratinocytes, and Langerhans’ islet cells of the pancreas as it plays a role in inflammatory response tissue homeostasis and giving a state of “immune privileged”, as the introduction of external antigens is tolerated with no immune or inflammatory response. PD-L1 is prompted as an inhibitory signal in inflammation acting upon immune, epithelial, and endothelial cells [15, 16].

Toll-like receptors (TLRs) in APCs affect PD-L1 expression through MEK/ERK (extracellular signal-regulated kinase) kinases activation, in addition to receptors of Interferon-gamma (IFN-γ) 1 & 2 via Jak (Janus kinase)/STAT (Signal transducer and activator of transcription)-mediated activation that can also influence MEK/ERK and PI3K/AKT pathway [17]. PD-1/PDL-1 ligation leads to SHP-1/SHP-2 recruitment to the ITSM with dephosphorylation of kinases e.g. CD3ζ, PKCθ, and ZAP70 leading to a general inhibition of T cell spreading out which results from PI3K-Akt and Ras-MEK-ERK cascade inhibition mostly through direct inactivation effect of PD-1of Ras &dephosphorylation of phospholipase Cγ [15, 18]. Dephosphorylation of Casein kinase 2 (CK-2), which is an SHP-2 target, causes uncontrolled activation of PTEN (PI3K-Akt signaling antagonist) [16, 17]. It was also suggested that CD28 receptor co-stimulation, maybe the main dephosphorylation target by SHP2 phosphatase [19]. PD-1/PD-L1 engagement modifies variable T cell activities including T cell proliferation deactivation, cytokine induction, survival, and other functions [20], the reaction between PI3K signaling and BCL-XL is a significant point of control where PD-1-inhibition of P13K decreases BCL-XL and endorses apoptosis [21] (Figure 1).

Figure 1.

Activation pathways of PD-1/PD-L1 expression.

The PD-1/PD-L1 interaction is critical for immune tolerance development, whether central or peripheral in primary or secondary lymphoid tissue respectively [22]. PD-1/PD-L1 knock-out in animal experiments causes autoimmunity with glomerulonephritis lupus-like arthritis and diabetes. While in humans, using antibodies against PD-1/PD-L1 leads to immune-related disorders such as endocrinopathy, colitis, and dermatoses [23, 24, 25]. A principal feature of T cell exhaustion, which is a marked weakening of effector T cell function, embraces the generation of several co-inhibitory pathways such as PD-1/PD-L1. Such impairment could be detected through apoptosis or inhibition of T cell development or production of regulatory T cells [26, 27]. The role of PD-1/PD-L1 is manifested in cases of T cell exhaustion, not only in chronic infection but also in cancer state [28, 29].

The greatest documented evidence of this inhibitory role in human immunity is derived from the usage of mediators to block the PD-1/PD-L1 pathway, an important target for immunotherapy in malignancy. Nevertheless, PD-1–PD-L1 interaction inhibition in patients suffering from malignancy causes anticancer immunity activation and autoimmune symptoms known as immune-associated opposing incidents [30].

3. PD-1/PD-L1 and cancer

3.1 PD-1/PD-L1 role in cancer

As PD-1/PD-L1 cardinal role is known in avoiding autoimmunity and preservation of normal peripheral tolerance, it is utilized by cancer cells in immune evasion eventually facilitating tumor growth, proliferation, and metastasis [6, 24].

Signaling of PD-1 in the tumor microenvironment, generated through interaction between cancer cells and non-transformed cells is an important player in the development and persistence of cancer through evasion of immune surveillance. PD-1 is markedly expressed in lymphocytes infiltrating a large number of cancers, also myeloid cells. While, PD-L1 is mainly conveyed on variable types of neoplastic cells such as melanoma, lung, renal and ovarian types. Moreover, PD-L1 expression can be up-regulated in different types of malignancy through carcinogenic signaling via aberrant PI3K-AKT activation or chromosomal amplifications and alterations, unrelated to inflammatory signaling in the microenvironment of malignancy [31, 32, 33].

Sometimes, PD-L1 expression is induced as a reaction to inflammation as an antitumor immune response via variable cytokines with IFN-γ being the most potent. IFN-γ causes PD-L1 activation and progression of ovarian neoplastic cells, while inhibition of IFN-γ receptor 1 can decrease PD-L1 expression in acute myeloid leukemia via the MEK/ERK and MYD88/TRAF6 pathways. Furthermore, IFN-γ prompts PKD2 (protein kinase D isoform 2), one of the PD-L1 regulator proteins. PD-L1 is also frequently expressed in cells of the microenvironment of tumors such as macrophages, dendritic cells, endothelial cells, and fibroblasts. PD-1/PD-L1 in tumor microenvironment endorses dysfunction and exhaustion of T cell, apoptosis, neutralization, and IL-10 secretion in a neoplastic mass generating resistance status against cancer cell destruction by a cytotoxic T cell (CD8+). Further infiltration by CD4+ regulatory T cells helps more suppression of effector immune response in tumors [31, 32].

The signaling of PD-L1 occurs via stimulation of PD-1which lead to pro-survival neoplastic signals induction helping anti-apoptotic effect via the cytoplasmic domain. In addition, PD-L1 protects neoplastic cells from the IFNs (I & II) cytotoxicity and CTL cytolysis. Furthermore, targeting PD-L1 decreases the activity of mTOR and neoplastic cell glycolytic metabolism without T cells [31, 34, 35].

PD-L1 positivity or negativity in tumor cells can be achieved via variable biological processes. T cells infiltrating the malignant tumor may produce surface expression of PDL-1 which can be lost in absence of T cells. Also, genetic proceedings inside the neoplastic cells could prevent PD-L1 expression. Thus, the neoplastic cell surface expression or absence of PD-L1 may implicate diverse functional significances and treatment allegations according to the causal expression mechanism [36].

Immunohistochemical expression of PD-L1 in neoplastic tissues shows that PD-L1 positive immune reaction may appear membranous or cytoplasmic. Trans membranous structure of PD-L1 suggests that the positive immune reaction may be related to the binding of PD-L1 antibody to a specific domain. While the cytoplasmic reaction may be related to the translocation of receptors onto the surface as a part of the immune response [37, 38, 39, 40].

Immunohistochemical expression of PD-L1 is still considered the merely broadly accessible, applicable, and cost-effective method for reviewing PD-L1 expression in cancer. Moreover, this method aids in recognizing patients who probably benefit from immunotherapy targeting PD-1/PD-L1. The FDA approval of such targeting therapy used trials depending on variable immunohistochemical platforms with different antibodies to evaluate PD-L1 expression on neoplastic cells, microenvironment immune cells, or both using specific scoring methods. These trials utilized particular PD-L1 inhibitors with particular assays and specific antibody reagents, thresholds, and protocols which should be standardized and validated. PD-L1 immunohistochemical procedure should be reproducible, both the staining technique and interpretation by pathologists which should be quality controlled starting from the tissue fixation and processing steps. Evolving applicable and reproducible scoring systems for PD-L1 is clinically important to identify patients who will probably benefit from targeted therapy. PD-L1 expression on both neoplastic and microenvironment immune cells has a greater association with clinical consequences in some neoplasms [41].

A recent systemic review and data analysis revealed the prognostic value of PD-L1. It shows high expression in solid malignancies which represents a bad prognostic feature regarding overall survival and progression-free survival [42]. The cytoplasmic expression and circulating tumor cells of PD-L1 were linked to better survival in thyroid carcinoma [43].

Blocking of PD-1 leads to suppression of transplanted myeloma cells growth in mice model while overexpressing PD-L1 in transplanted cells of mice model leads to the neoplastic establishment, load, and invasiveness which were reversed by using antibodies against PD-1/PD-L1. Thus, down-regulation of PD-1 and/or PD-L1 enriches T-cell activation against malignant cells which is the base for immunotherapy [33, 44].

3.2 PD-1/PD-L1 as a targeted therapy

Enhanced cancer suppression can be perceived when engaging different methodologies of PD-1/PD-L1 signaling disturbance, such as blocking using an antibody against PD-L1, DNA vaccination of PD-1 extracellular region, and injection of neoplasm-specific clones of T cells. The use of several immunotherapy approaches in combination may increase the therapeutic outcome [43, 44, 45].

The regulation of neoplastic cells’ expression of PD-L1 includes signaling pathways (MAPK, PI3K/Akt), transcription factors (e.g., Hypoxia-Inducible Factor 1, STAT3, Nuclear Factor kappa B, Transforming Growth Factor beta, GATA-3, and T-bet), and epigenetic and micro RNAs regulation [11].

In clinical practice, blocking of PD-1/PD-L1 causes inhibition of immune checkpoints which provides long-term responses, and as such the blocking antibodies are approved to treat solid and hematologic neoplasms. As well, the cytoplasmic PD-L1 knockdown using particular RNAs could benefit tumor immunotherapy [46].

The PD-L1/PD-1 blocking antibodies are the mainstay of immunotherapy due to improved survival and their clinical effectiveness in various malignancies such as non-small cell lung carcinoma (NSCLC) [47, 48]. Neoplasms showing an increased capacity for mutation and antigenicity, e.g., high microsatellite instability (MSI) and mismatch repair deficiencies (dMMR) are good targets for PD-1 blocking therapies. Numerous elements play significant roles as a determinant of clinical response to blocking PD-L1/PD-1 pathway such as neoplasm mutation load densities of immune cells and tumor microenvironment types of cells, PD-1/PD-L1 level of expression, and cytokines [49, 50].

Immunotherapy is considered a safe treatment compared with other strategies such as chemotherapy, irradiation, and surgery, as the mechanism involves augmentation of self-immunity against malignancy. Moreover, as a checkpoint inhibitor, it is extremely precise to a targeted cell with fewer side effects and keeps antigenic memory of neoplasm. However, it has been observed that associated toxicities with PD-1 blocking antibodies is lower than the associated toxicity with other immunotherapies, e.g., CTLA-4 blocking agents [45, 47, 51, 52, 53].

PD-1/PD-L1-induced opposing events related to immunity are one of the disadvantages of this type of therapy. It can induce side effects related to immunity in different organs such as the endocrine, pancreas, skin, gastrointestinal tract, liver, and renal system. Furthermore, such antibodies induced lethal xenogeneic hypersensitivity reactions in an experimental model of breast cancer after repeated administration. It is worth mentioning that PD-1 blocking-related pneumonitis is a significant side event mostly observed in NSCLC patients. Other systemic side effects, e.g., cardiac arrhythmia and even heart failure due to myocarditis have also been reported [30, 54, 55]. Clinically, subcutaneous or intravenous route of administration in this type of therapy is considered as one of the drawbacks, especially with humble penetration of neoplastic tissue [56].

PD-1 blocking mediators are associated with increased rates of recurrence and progression of the disease, nevertheless, local therapy can produce long-term survival without progression in some of these patients [57]. PD-L1 expression is used as an authenticated and main biomarker for the prediction of therapy; still, this biomarker alone, due to tumor heterogeneity is considered insufficient for defining patients who can benefit from PD-1/PD-L1 blocking therapy. Then again, PDL-1 single nucleotide polymorphisms (genetic copy number gains) have been suggested to help predict treatment responders, [52, 58, 59], especially in lymphoma [60, 61]. This is because de-glycosylation of the natural heavily glycosylated surface PD-1 molecules by enzymes during immunohistochemistry increases antibody binding ability of anti-PD-L1, thus increasing the intensity of signals leading to better outcome prediction [62].

The prediction of patient response is an important issue in PD-1/PD-L1 blocking therapy as only one to two-thirds of patients show resistance to treatment due to heterogeneity [52, 63]. The absence of neoplastic antigens, neoantigens or gene mutations, dysfunction of T cell, expression of PD-L1 and tumor microenvironment, noncoding RNA, and gut microbiome are also underlying factors serving as mechanisms of resistance to PD-1/PD-L1 blocking treatment [64].

Furthermore, PD-1 blocking therapy is largely costing more than other immunotherapy treatments and original lines of treatment, especially in old patients with low income and there is a debate regarding the cost-benefit relationship in combined therapy [65]. Using such an immunotherapy line of treatment in patients with immune disease whether hyperactive or autoimmune or hypoactive or even deficient immunity is considered a challenge, particularly with the toxicity effect of such therapy [53, 66].

3.3 Improving the PD-1/PD-L1 blocking efficacy

The regulatory mechanisms and pathways of PD-L1 expression have been widely investigated to understand side effects and deficient responses in some patients. The combination approaches were introduced to concurrently enhance several cancer-immunity processes, eliminate brakes of immunosuppression, and coordinate an immune-enhanced neoplastic microenvironment [53, 67].

Combination lines of treatment, such as combining anti-PD-1/PD-L1 with radiotherapy, chemotherapy, other immune checkpoint inhibitors or targeted therapy, interferon genes agonists stimulator, transplantation/manipulation of microbiome, epigenetic or metabolic modulators may produce better treatment response. Furthermore, agents containing both PD-1 and PD-L1 targeting antibodies may also provoke a potent effect. Regulation of PD-L1 expression in the tumor microenvironment through medical treatment or regulation of genes expands the -PD-1/PD-L1 therapy effect [68, 69, 70].

Possible small-molecule mediators were suggested to be used for targeting PD-L1 which may help overcome the restrictions of monoclonal antibodies used in blocking PD-1/PD-L1. Preclinical investigations suggest that the combination of these small-molecule mediators with other immune checkpoints targeting agents may cause augmented antineoplastic action or use such small molecules to up-regulate PD-L1 and elevate the blocking efficacy [55, 71]. Moreover, prodrug nanoparticles conjugated with anti-PD-L1 peptide were suggested to be used to help inhibit neoplastic growth with minimum side events [72].

Other effective antineoplastic agents, e.g., antiangiogenic mediators, and immunogenic cell death inducers combined with immune checkpoint inhibitors may help as a preventive therapeutic method in improving blocking agents’ efficacy [73, 74].

4. Conclusion

Taking into account the various potential strategies for successful PD-1/PD-L1 checkpoint inhibition such as blocking using antibodies against PD-L1, DNA vaccination of PD-1 extracellular region, injection of neoplasm-specific clones of T cell, with understanding mechanisms of action is important in clinical practice. A deep consideration of the mechanisms of resistance, whether cellular or molecular will benefit patients and improve therapeutic approaches. The combination approaches of immunotherapy or with other lines and strategies of therapy were introduced, also, small-molecule mediators and prodrug nanoparticle conjugation were suggested to be helpful in cases of anticipated resistance. The future perspective of combination therapy and investigation of predictive biomarkers will provide an important pathway for cancer patient care in cases treated with PD-1/PD-L1 checkpoint inhibition.