During the transcription and expression of Pdcd1, the gene encoding PD-1, two conserved regions (CR-B and CR-C) located upstream of the transcription start site (TSS) of the Pdcd1 gene play an important role (16). Researchers found that there are regulators in both transcriptional regulation and epigenetic regulation of PD-1 expression that are closely associated with these two conserved regions. In terms of transcriptional regulation, the CR-B region contains binding sites for activator protein 1 (AP-1) and the negatively regulated transcription factor T-bet.AP-1 is a class of four sub AP-1 is a class of transcriptional activators consisting of four subfamilies (Fos, Jun, ATF and Maf) (17). It is involved in various cellular processes, including cell differentiation, proliferation and apoptosis, by regulating the expression of related target genes (18). Xiao et al. (19) found a large amount of AP-1 subunit protein c-Fos in complex with JunB on tumor-infiltrating T cells, and found that the latter bound to the AP-1 binding site in the CR-B region, resulting in upregulation of PD-1 expression. T-bet was the first regulatory factor identified to have the ability to repress Pdcd1 transcription. Further studies have demonstrated that it can negatively regulate PD-1 transcription by binding directly to the corresponding site in the CR-B region after stimulation by TCR signaling (20). However, the researchers found that PD-1 expression in CD8+ T cells was only slightly increased in T-bet knockout mice after viral infection compared to the comparison group, which also suggests that T-bet may need to synergize with other factors to co-inhibit PD-1 expression. Compared with the CR-B region, the CR-C region has a richer set of regulatory sites, including nuclear factor of activated T-cells cytoplasmic 1 (NFATc1) binding sites, IFN-stimulated response element (ISRE), nuclear factor-κB (NF-κB) binding site and forkhead transcription factor 1 (FoxO1) binding site (21). The persistence and strength of the TCR signaling pathway affects the expression of PD-1 in CD8+ T cells, where the TCR/Calcineurin/NFATc1 pathway is considered to be one of the major initiating upstream signaling pathways that activate PD-1 transcriptional expression. The transcription factor NFATc1 binds to its counterpart element within the CR-C region upon stimulation by TCR signaling to initiate PD-1 transcription, which in turn promotes PD-1 expression (16). ISRE is a PD-1 regulatory element located within the CR-C region, and its activation is closely associated with the generation of the IFN-stimulated gene factor 3 (ISGF3) complex. When IFN-α activates the JAK/STATs signaling pathway, interferon response factor 9 (IRF9) and STAT1-STAT2 heterodimers combine to form ISGF3 and then bind to ISRE to promote PD-1 expression on CD8+ T cells and macrophages (22). And for CD8+ T cells alone, Terawaki et al. (23) found that treatment of T cells with IFN-α alone did not promote PD-1 expression, but when activated via the TCR signaling pathway, treatment of T cells with IFN-α enhanced PD-1 expression. The TCR/Calcineurin/NFATc1 signaling pathway was able to induce T cell transcription of Pdcd1 in macrophages, but not in macrophages. Bally et al. (24) found that during activation of macrophages, Pdcd1 transcription was activated by NF-κB, and the p65 subunit of NF-κB was able to bind to the corresponding element within the CR-C region and upregulate PD-1 expression. In the early stages of chronic infection, NFATc1 is a regulator that activates Pdcd1 transcription in CD8+ T cells. Agnellini et al. (25) found that NFATc1 is unable to enter the nucleus of CD8+ T cells after chronic infection is established, when Pdcd1 transcription is activated by FoxO1. DNA methylation of the gene promoter region represses gene transcription (26). In terms of epigenetic regulation, CR-B and CR-C were fully methylated in naive T cells that did not express PD-1, whereas during the differentiation of naive T cells to effector CD8+ T cells after viral infection, PD-1 expression levels were continuously upregulated and the methylation levels of these two regions gradually decreased, with a significant negative correlation between them (27). This also suggests that changes in methylation in the CR-B and CR-C regions have an important regulatory role on PD-1 expression.

Outside of these two major regulatory regions there are also a number of role elements involved in the regulation of PD-1 expression, such as those located at the B lymphocyte induced maturation protein-1 (B lymphocyte-1, Blimp-1) binding site, signal (B lymphocyte induced maturation protein-1, Blimp-1) binding sites, signal transducers and activators of transcription (STATs) binding sites, recombination signal binding protein-Jk (RBPJk) binding sites associated with the Notch signaling pathway, and Blimp-1 is a transcriptional repressor that functions similarly to T-bet, and when activated by T cells, Blimp-1 binds to the binding site between CR-C and CR-B and directly affects the inhibition of Pdcd1 transcription. In addition to this, Blimp-1 can also inhibit the expression of NFATc1 (the transcriptional activator of Pdcd1). And through this feed-forward loop can rapidly shut down the transcription of Pdcd1 thereby inhibiting the expression of PD-1 (28). STAT3 (or STAT4) is able to bind to the 5′ and 3′ distal sites of the TTS of the Pdcd1 gene and directly regulate the transcriptional expression of PD-1 in activated CD8+ T cells (29). In addition, Mathieu et al. (30) found the presence of RBPJk binding sites upstream of CR-C, which upon binding were able to activate the transcriptional expression of Pdcd1. Two other important Pdcd1 regulatory elements are the isolators (insulators) located on either side of Pdcd1 (-25.7 kb and +17.5 kb). They are two terminals of Pdcd1 cis-regulation,which bind the corresponding transcription factor (CTCF) upon NFAT stimulation respectively, thereby altering the topology of the regulated gene and preventing activation of unrelated genes by enhancers (29) ( Table 1 ).

PD-L1 expression in tumor cells is not only regulated by pro-oncogenic signaling pathways, but also influenced by multiple factors in the tumor microenvironment (TME) and multiple intracellular enzymes (37). At present, the mechanisms regarding the regulation of PD-L1 in cancer can be grouped into four categories, namely regulation through genomic alterations and rearrangements, signaling pathways, miRNAs and post-translational modifications.

Intra-tumor gene mutation and amplification are associated with the regulation of PD-L1. When located on chromosome 9p24.1 After amplification and translocation of CD274, the gene encoding PD-L1, in some tumors (primary mediastinal large B lymphocytoma, non-small cell lung cancer, squamous cell carcinoma and EBV-positive gastric cancer) there may be an increase in PD-L1 expression levels (38, 39).

Oncogenic signaling pathways in tumors are not only involved in the regulation of tumorigenesis and progression, but also in the regulation of PD-L1 expression to assist tumor cells in generating immune escape ( Figure 1 ). This regulation is mainly involved through signaling transcriptional regulators, pathway effector molecules and upstream receptors. Many transcriptional regulators are involved in the direct transcriptional regulation of PD-L1, for example, Myc can promote PD-L1 expression by binding to the promoter of the PD-L1 gene (40). It has also been reported that MYC binding to the PD-L1 promoter leads to downregulation of PD-L1 expression, which enhances tumor immunocidal capacity (41). Nuclear transcription factor-κB (NF-κB) is a transcription factor widely expressed in various cell types, which can be activated by multiple inflammatory signaling pathways and oncogenic mutations. Tumor necrosis factorα (TNF-α) is a type II transmembrane protein that has a strong killing effect on tumor cells and is also one of the important activators of NF-κB. Asgarova (42) found that when lung cancer cells undergo epithelial- mesenchymal transition (EMT) during treatment with TNF-α resulted in NF-κB recruitment at the promoter of PD-L1 and upregulation of PD-L1 expression. It was shown that RelA, a subunit of NF-κB, is able to bind to the PD-L1 promoter region, further suggesting that NF-κB can be directly involved in the regulation of PD-L1 expression (43). STAT3 is a transcription factor that plays a key role in the production of immunosuppressive factors and promotion of cancer cell proliferation in the tumor microenvironment. Atsaves (44) STAT3 is a transcription factor that plays a key role in the production of immunosuppressive factors and promotion of cancer cell proliferation in the tumor microenvironment. Hypoxia is a common feature of most solid tumors, and hypoxia activates the expression of downstream genes by promoting the expression of hypoxia-inducible factors (HIF-1α and HIF-2α). HIFs can directly bind to and induce transcription of the hypoxia response element (HRE) of the PD-L1 promoter region (45). Thus overexpression of HIF-1α leads to upregulation of PD-L1 expression. In addition to binding to the promoter region, AP-1 is able to bind the enhancement region of the first intron of PD-L1 to regulate the transcription of PD-L1 (46). Cyclin-dependent kinase 5 (CDK5) is a non-classical cyclin protein kinase capable of promoting several processes such as angiogenesis, apoptosis, and vesicle transport.IRF2 and IRF2BP2 are PD-L1 transcriptional repressors and CDK5 can regulate PD-L1 transcription by repressing the expression of both and thereby regulating PD -L1 transcription (47). In addition, activation of MAPK signaling pathway and PI3K signaling pathway promotes PD-L1 expression by activating transcription factors c-Jun and Akt respectively.

MicroRNAs (miRNAs) are a class of small non-coding single-stranded RNAs consisting of 20-22 nucleotides, which cause degradation or translation inhibition of target gene mRNA by targeting the untranslated region (3’-UTR) at the 3’ end of the mRNA (48). Several reports have shown that mi RNA can miRNA regulates PD-L1 expression in tumors by targeting the 3’-UTR of PD-L1 mRNA, including gastric cancer, pancreatic cancer, ovarian cancer, non-small cell lung cancer, and leukemia (49–52). Among these miRNAs, most of them repress PD-L1 expression by binding to PD-L1 mRNA, such as: miR-93, miR-142-5p, miR-138-5p, miR-106b, miR-200, miR-217, miR-570, miR-152, miR-15a, miR-17-5p, miR-193a and miR-16. And Zhu et al. (53) found that miR-130b, miR-20 and miR-21 indirectly promoted PD-L1 expression in colon cancer by inhibiting PTEN expression.

Post-translational modification (PTM) refers to post-translational chemical modifications of proteins, including phosphorylation, ubiquitination, acetylation, glycosylation and methylation. Precursor proteins are inactive and often undergo a series of post-translational processing to become functional mature proteins.PD-L1 is modified by different post-translational modifications that affect its localization, stability, and pathological and physiological functions (54). Li et al. (55) found that glycogen synthase kinase 3 beta (GSK3β) can directly bind to the C-terminal structural domain of PD-L1, catalyze the phosphorylation of the T180 and S184 sites of PD-L1 and then be recognized by Beta-transducin repeats-containing proteins (β-TRCP), and ultimately promote the polyubiquitination and degradation of PD-L1. Ubiquitination is the process by which ubiquitin molecules sort intracellular proteins by a series of specific enzymes, from which target protein molecules are selected and specifically modified. Zhang et al. (56) found that CDK4 can negatively regulate PD-L1 by mediating SPOP protein phosphorylation to indirectly ubiquitinate PD-L1 for degradation. While Chemokine-like factor MARVEL transmembrane domain-containing family (CMTM) family members CMTM6 and CMTM4 not only enhance PD-L1 protein half-life and inhibit PD-L1 ubiquitination, but also protect PD-L1 from lysosomal-mediated degradation. N-glycosylation is key protein modification that determines protein structure and function (especially membrane protein function). The extracellular region of PD-L1 (N219, N200, N192, N35) is a glycosylation binding site, and glycosylation prevents GSK3β-induced leading to PD-L1 phosphorylation and ubiquitination, which can play a role in stabilizing PD-L1 (57) ( Table 2 ).

Tumor immunotherapy represented by PD-1/PD-L1 monoclonal antibodies has opened a new era of tumor treatment. However, immunotherapy has a high rate of drug resistance compared with molecular targeted therapy and chemotherapy, which is a problem that cannot be ignored in clinical practice (84). The development of PD-1/PD-L1 antibody resistance is a complex, dynamic and interdependent process, with many intrinsic and extrinsic tumor cell factors associated with it ( Figure 2 ). Resistance can be classified into secondary and primary resistance according to the time of its emergence. Secondary resistance refers to the emergence of resistance after the initial treatment has been effective in controlling tumor progression. Primary resistance, on the other hand, exists prior to exposure to the drug and prevents the drug from impeding tumor progression (85). PD-1/PD-L1 resistance has been shown to be associated with PD-1/PD-L1 expression levels on the cell surface, the absence of first and co-stimulatory signals, the tumor microenvironment, and epigenetic modifications.

Although PD-1 or PD-L1 inhibitors can block the linkage of both, they cannot affect the expression of PD-1/PD-L1 on the cell surface, and the high expression of PD-1 on the T cell surface may be related to PD-1/PD-L1 inhibitor resistance. In the tumor microenvironment tumor cells and antigen presenting cells (APCs) upregulate PD-L1 expression through 2 pathways, adaptive immune tolerance and intrinsic immune tolerance, to evade immune surveillance, and PD-L1 upregulation is also a prerequisite for the efficacy of anti-PD-1/PD-L1 monoclonal antibodies. Juneja et al. (86) have demonstrated through mouse experiments that PD-L1 expression on tumor cells and immune cells levels play a key role in the efficacy of PD-1 antibodies. Therefore, the expression of PD-1/PD-L1 can be influenced by modulating some of the regulators described above, thereby improving the response to PD-1/PD-L1 inhibitors in tumor or non-tumor patients.

Unlike normal cells, tumor cells possess immunogenicity because they mutate to induce the expression of specific antigens on the cell surface. Tumor antigens are acquired by antigen presenting cells (APCs) and presented to T cells. Activated T cells then play a therapeutic role. The most direct reason for the failure of PD-1/PD-L1 antibodies to treat tumors is the inability of T cells to recognize them due to the lack of tumor antigens. Sade-Feldman et al. (87) found that mutations or deletions of beta-2-microglobulin (B2M) resulted in the loss of MHC I expression and impaired antigen recognition, resulting in the inability of cells to recognize tumor cells, which in turn led to drug resistance. Tumors can reduce the number of APCs by secreting inhibitory factors such as vascular endothelial growth factor (VEGF) and interleukin-10 (IL-10). When APC is abnormal, it also leads to impaired signaling, which in turn leads to abnormal T-cell recognition. In addition to the first signal, T cells require a second signal, namely co-stimulatory signals, to exert their immune action. After evaluating the effects of CD40 agonists using a chronic infection model, Xu et al. (88) found that CD40 agonists could enhance the therapeutic effects of PD-1 inhibitors by transforming inactivated PD-1-expressing cytotoxic T lymphocytes (CTL) through activation of the mTORC1 pathway. This also suggests that co-stimulatory signaling stimulators can enhance T-cell function. And when co-stimulatory signaling is absent, the efficacy of PD-1/PD-L1 inhibitors is reduced and drug resistance develops.

The tumor immune microenvironment is dynamically regulated by the interaction of tumor tissue and associated immune cells, providing a chronically inflammatory and immunosuppressive environment for tumor survival and progression. In addition to tumor cells, components that may be associated with primary or acquired drug resistance include tumor-infiltrating lymphocytes, CD8+ T-cell depletion, tumor-associated immunosuppressive cells, and other suppressive immune checkpoints. Tumor infiltrating lymphocytes (TIL) are the effector cells of the immune response and the effect of antitumor immunity is related to their activity and number. Tumeh et al. (89) found that the presence of CD8+ T cells in the tumor was a prerequisite for tumor shrinkage after treatment with anti-PD-1/PD-L1 monoclonal antibodies in metastatic melanoma, and that chemokine expression played a key role in the migration of T cells from the circulatory system into the tumor. When the number of tumor-infiltrating lymphocytes is reduced the effect of immunotherapy can be diminished or even lead to drug resistance. In the tumor setting, CD8+ T cells show upregulation of PD-1 expression in response to sustained antigen stimulation, and PD-1 interacts with PD-L1 to form a depleted T cell phenotype. Anti-PD-1/PD-L1 monoclonal antibodies were able to reverse PD-1 moderately expressed failing CD8+ T cells, but were ineffective against severely failing CD8+ T cells with high PD-1 expression (90). This suggests that the key to reversing PD-1/PD-L1 antibody resistance may lie in the ratio between the two. Tumor-associated immunosuppressive cells located in the tumor stroma include bone marrow-derived suppressor cells (MDSCs), regulatory T cells (Tregs) and M2 macrophages. They diminish the effect of anti-PD-1/PD-L1 monoclonal antibodies by suppressing antitumor effector T cell function. MDSCs are major regulators of immune responses in various pathological conditions, and they can promote tumor invasion and metastasis by promoting angiogenesis. In addition, MDSCs present in the tumor microenvironment reduce the effectiveness of immunotherapy (91). Tregs suppress effector T cell function by secreting suppressive cytokines (TGF-β, IL-35, and IL-10). Ngiowet et al. (90) found that Tregs promote CD8+ T cell depletion and the CD8+/Tregs ratio can be used as a predictor of anti-PD-1 monoclonal antibody efficacy. And M2 macrophages are tumor-associated macrophages (TAMs) with pro-cancer properties. Clearance of these cells may enhance the effect of anti-PD-1/PD-L1 monoclonal antibodies. In addition to PD-1/PD-L1, other immune checkpoints such as TIM-3, CTLA-4, LAG-3 and IDO are also involved in regulating the immune response, and they also have an impact on PD-1/PD-L1 antibody efficacy (92). For example, tumor recurrence after PD-1/PD-L1 antibody treatment may be the result of increased TIM-3 expression in T cells. In contrast, LAG-3 monoclonal antibody can restore the body’s anti-tumor immune activity by reducing the number of bone marrow-derived suppressor cells (MDSC), inhibiting the activity of Treg cells in the tumor microenvironment and increasing the number of activated CD8+ T cells (93).

In terms of epigenetic modifications, the epigenetic factors histidine-lysine N-methyltransferase (enhancer of zeste homolog 2, EZH2) and DNA methylation may be important mechanisms mediating immunotherapy resistance. Emran et al. (94) found that DNA hypermethylation inhibits the endogenous interferon response required to recognize cancer cells, leading to immune suppression. While hypomethylation also leads to increased expression of the inhibitory cytokines VEGF, IL-6 and PD-L1 and thus affects the efficacy of immunotherapy. In contrast, DNA methyltransferase inhibitors can reverse immunosuppression by affecting antigen processing and delivery and enhancing the expression of other immune-related genes, co-stimulatory molecules, tumor-associated antigens and chemokines (95). In addition, DNA methylation has been found to cause T-cell depletion, and by reversing the depleted state of T cells can promote the restoration of T cells to their immune function (96).

CTLA-4 is a transmembrane protein on the surface of T cells, expressed on the surface of activated CD8+ T cells, CD4+ T cells and Treg cells (99). And it is a key inhibitory receptor affecting T cell function. CTLA-4 inhibitors efficiently bind CTLA-4 molecules, reducing the number of CTLA-4 molecules bound to B7 molecules, and inhibiting T cell activation by reducing the production of negative regulatory signals, enhancing T cell activation and the growth and infiltration of tumor-differentiated T cells in the tumor microenvironment, thereby achieving antitumor effects of the immune system. Some CTLA-4 inhibitors have been approved by the Food and FDA for clinical use with promising results (100). And representative drugs include Ipilimumab and Tremelimumab. PD-1 acts in the late stage of T-cell activation, after T-cell migration into the tumor microenvironment, while CTLA-4 acts in the early stage of T-cell activation (101). Beavis et al. (102) found that CD4+Foxp3-T cells are effector T cells with anti-tumor functions, and increasing the number of CD4+Foxp3-T cells in tumor tissue can improve the effectiveness of tumor treatment. Both CD8+ T cells and CD4+Foxp3-T cells could be activated when CTLA-4 and PD-1 were blocked simultaneously. This provides a rationale for the use of CTLA-4 in combination with PD-1/PD-L1 inhibitors for cancer treatment. This combination approach has become the most intensively studied as well as the most widely used immune combination regimen (103). Daniel et al. (104) evaluated the efficacy of Pembrolizumab plus Ipilimumab in melanoma patients after failure of anti-PD-1/PD-L1 therapy and showed that 29% of patients achieved a confirmed response (including 21.4% partial responses and 7.2% complete responses). This demonstrates the significant antitumor activity of the combination. A study of long-term follow-up of patients with advanced melanoma treated with Nivolumab Plus Ipilimumab showed that these patients had a 3-year Overall Survival (OS) of 63% (105). This also demonstrates the effectiveness and durability of the combination of the two. Although combination therapy showed multiple advantages, in terms of safety, it had a higher incidence of treatment-related adverse events than chemotherapy (24.5% vs. 13.9%), and discontinuation due to treatment-related adverse events was more common (18.1% vs. 9.1%) (106). In a meta-analysis of solid tumors, the probability of immunotherapy-related adverse reactions following treatment with nivolumab in combination with ipilimumab was higher than with nivolumab alone. of these adverse reactions, the incidence of grade 3+ gastrointestinal toxicity (9.9% vs. 1.2%) and hepatotoxicity (10.1% vs. 1.3%) were significantly higher (107). In addition, a study of non-small cell lung cancer treatment showed that the incidence of grade 3 or higher treatment-related adverse events was 33.8%, 22.9%, and 14.9% in the chemotherapy group, the Durvalumab combined with Tremelimumab group, and the Durvalumab group, respectively (108). Different dosing sequences, drug doses, and dosing intervals in different tumors can affect the frequency and severity of adverse events. Sen et al. (109) demonstrated that the incidence of immune-related adverse events (irAEs) increased with increasing doses of immune checkpoint inhibitors, but that disease control rate (DSR), OS, and PFS did not improve. This also justifies the low-dose immune checkpoint inhibitor regimen. According to the results of the Checkmate012 study, low-dose combinations may hold promise for reducing the incidence of adverse events and improving clinical efficacy (103). In a retrospective analysis of 134 unselected stage IV solid cancer patients, Kleef et al. (110) found that the use of off-label low doses of nivolumab plus ipilimumab combination did not compromise efficacy and that the post-treatment emerged with a safer irAEs profile. Research on such combinations is currently in full swing, and in the future the optimal dosing order, dosing interval, and dosing review rate for different tumors may be answered.

T-cell immunoglobulin adhesion protein molecule 3, a member of the Tim family, is a type I transmembrane glycoprotein.TIM-3 is widely distributed on the surface of T cells, regulatory T cells, and innate immune cells (DC cells, NK cells, monocytes). It was first found to be expressed at high levels in “depleted” T cells of HIV-infected patients and was later shown to be an important player in T cell depletion (111). TIM-3 can lead to T cell depletion and contribute to tumor immune escape, invasion and metastasis, thus affecting patient outcome and prognosis. It is worth mentioning that although TIM-3 is considered to be a suppressor receptor, one study found that TIM-3 can also play a role in activating T cells (112). Sakuishi et al. (113) found that on most tumor infiltrating lymphocytes (TILs) in mice bearing solid tumors,and there was co-expression of PD-1 with TIM-3. They also found that the combined use of anti-PD-1 and anti-TIM-3 significantly slowed down tumor growth, demonstrating the effectiveness of combined use of PD-1 and TIM-3 inhibitors in controlling tumor growth. Currently, TIM-3 inhibitors such as TSR-022, MBG453, Sym023, LY3321367, INCAGN-02390, and BGB-A425 have entered clinical trials, and researchers need to conduct numerous trials before they can conclude whether their combination with PD-1/PD-L1 inhibitors in clinical use can meet expectations.

LAG-3, also known as CD223, is a type I transmembrane protein that is also a member of the immunoglobulin superfamily. It is mainly expressed on the surface of activated CD4+ T cells and CD8+ T cells, but also on the surface of other immune cells such as B cells, natural killer cells and dendritic cells (114). However, under physiological conditions, naive and normal T lymphocytes express only low levels of LAG-3. After tumor antigen stimulation, the level of LAG-3 expression in lymphocytes increases substantially (115). At the same time, its expression level was positively correlated with tumor development, suggesting that interruption of LAG-3 may enhance anti-tumor immunity.LAG-3 not only inhibits T cell activation and proliferation, but also promotes the activity of regulatory cells (Tregs). This allows LAG-3 antibodies to simultaneously restore T-cell function and suppress Treg cell activity. In contrast, according to previous studies antibodies against PD-1 can only restore T-cell function (116). This has led researchers to see the value of the research behind it. Woo et al. (117) found extensive co-expression of LAG-3 and PD-1 on tumor-infiltrating CD8(+) and CD4(+) T cells, and used anti-LAG-3/PD-1 dual antibodies to treat mice already resistant to single antibody treatment, and found that this combination cured most of the mice. Bottai et al. (118) found that PD-1 and LAG-3 were simultaneously expressed on tumor-infiltrating lymphocytes in approximately 15% of triple-negative breast cancer patients, providing an opportunity to evaluate anti-LAG and anti-PD-1/PD-L1. BMS-986016 (Relatlimab), the first IgG4 monoclonal antibody developed to target LAG-3, has been clinically tested in a variety of cancers and has been shown to be effective in combination with Nivolumab in advanced melanoma patients treated with anti-PD-1/anti-PD-L1 therapy. Sym022, MK-4280, REGN3767, TSR-033 and other LAG-3 inhibitors are in early clinical trials. Therapeutic combinations of different types of LAG-3 monoclonal antibodies in combination with PD-1/PD-L1 inhibitors will be used for cancer treatment in the future.

Indoleamine-2,3-dioxygenase (IDO) is an enzyme that converts tryptophan to kynurenine, which can affect cell survival. Thus, T cell proliferation is inhibited when tryptophan is lacking in the tumor microenvironment (119). Dill et al. (120) found that about 70% of TNBC L1 + breast cancer cells also expressed IDO, suggesting that IDO-related T cell damage may be the mechanism of anti-PD-1/PD-L1 immunotherapy drug resistance. Therefore, TNBC patients expressing IDO can be treated with PD-1/PD-L1 inhibitors combined with IDO inhibitors. A phase Ib clinical trial showed that 9% of patients treated with Atezolizumab combined with IDO inhibitor Navoximod reached partial response (PR) and 17% reached stable disease (SD). Most of the treatment-related adverse events were grade 1 and 2, of which the main ones were rash (22%) and fatigue (22%) (121). In addition to the these more commonly used immune checkpoint inhibitors mentioned above, TIGIT inhibitors, B7-H3 monoclonal antibodies, VISTA inhibitors, and other combinations with PD-1/PD-L1 inhibitors are also being evaluated in clinical trials.

Tumor vasculature is highly abnormal compared to normal tissue vasculature, and the main cause of vascular abnormalities is the overexpression of VEGF. Immunosuppression and angiogenesis are two processes that are closely related during tumor development. Vasculature in tumors can exert immunosuppressive effects through various mechanisms, the four most important of which are: (1) promoting the recruitment of immunosuppressive cells (tumor-associated macrophages, myeloid-derived suppressor cells, regulatory cells) and proliferation (139, 140). (2) Inhibit the proliferation, transport and function of cytotoxic T lymphocytes (141). (3) Promoting abnormal tumor angiogenesis, leading to low pH and hypoxia in the tumor microenvironment, which subsequently causes immunosuppression systemically and locally (142, 143). (4) Inhibit antigen presentation and dendritic cell maturation, thereby hindering T cell activation (144, 145). PD-1/PD-L1 inhibitors can normalize tumor vasculature by promoting γ-interferon production and activating effector T cells, thereby enhancing the killing function of effector T cells and the efficacy of VEGF inhibitors. The combination of PD-1/PD-L1 inhibitors and VEGF inhibitors has been extensively studied in patients with various cancers. In 2019, the combination of pembrolizumab and axitinib was approved as a first-line agent in advanced renal cell carcinoma (146). Uemura et al. (147) evaluated two regimens for the treatment of advanced renal cell carcinoma, (avelumab + axitinib) and sunitinib, and found that patients treated with avelumab + axitinib had higher objective remission rates (ORR) and progression-free survival (PFS). However, it has also been shown that high doses of VEFG inhibitors in combination with PD-1/PD-L1 inhibitors can directly disrupt tumor vasculature, which can lead to more severe hypoxia and thus exacerbate tumor progression (148). A study of untreated patients with advanced NSCLC treated with an anti-VEGFR monoclonal antibody in combination with Pembrolizumab showed an incidence of grade 3+ treatment-related adverse events of 42.3%, with hypertension (15.4%) and acute myocardial infarction (7.7%) being the two most common adverse reactions (149). Results from another study comparing the Atezolizumab group with the Atezolizumab combined with Bevacizumab group showed that the incidence of grade 3+ TRAEs was 5% and 20% in the two groups, respectively. Of these, the most common adverse reactions in the combination group were hypertension (5%) and proteinuria (3%) (150). Currently, clinical data on the combination of PD-1/PD-L1 inhibitors with VEGF inhibitors for the treatment of cancer are scarce, and future studies will focus mainly on determining the optimal combination and dose of VEGF inhibitors and PD-1/PD-L1 inhibitors for better anti-cancer efficacy.

EGFR belongs to the tyrosine kinase receptor (ErbB) family, a cell surface receptor encoded by the HER1 (ErbB1) gene. binding of the extracellular portion of EGFR and extracellular ligands allows EGFR monomers to form dimers with other EGFR molecules or other ErbB family protein receptors. Dimer phosphorylation promotes tumor cell proliferation, differentiation and migration through activation of the downstream signaling pathways RAS-PI3K-PTEN-AKT-mTOR and RAS-RAF-MEK-ERK (151). Mutations and excess phenotypes or mutations of EGFR are associated with aggressive tumor behavior. including treatment resistance and metastasis of tumor cells. Its high expression is particularly evident in breast cancer (152, 153). However, researchers have found that its effectiveness in treating cancer as a single agent does not meet expectations. Akbay et al. (76) found that mutations in EGFR in NSCLC cause high expression of PD-L1, allowing tumor cells to evade the immune system through the PD-1/PD-L1 pathway. Consistent with these findings, Li et al. (55) found that EFGR inhibitors such as lapatinib, erlotinib and gefitinib can reduce epidermal growth factor-induced PD-L1 expression and thus achieve anti-tumor effects. These findings also illustrate the feasibility of combining EGFR inhibitors and PD-1/PD-L1 inhibitors for cancer treatment. Sugiyama et al. (154) used an anti-PD-1 monoclonal antibody in combination with an EGFR inhibitor in a mouse model of lung cancer and found its efficacy to be better than either treatment alone. However, in a retrospective study, researchers found that 15% of patients who used PD-L1 antibodies followed by the combination of oxitinib experienced immune-related adverse effects. And the shorter the interval between the use of both, the higher the probability of adverse reactions (155). A phase I trial evaluating Atezolizumab in combination with Erlotinib in patients with NSCLC showed a 39% incidence of grade 3 or higher treatment-related adverse events among patients, with fever, rash, and elevated alanine transaminases each accounting for 7% of the most common (156). Therefore, the combination of PD-1/PD-L1 inhibitors with egfr inhibitors needs to be used with caution while avoiding the occurrence of adverse reactions.

PARP is a poly ADP ribose polymerase present in eukaryotic cells, which is involved in the repair of dna damage and the regulation of apoptosis (157). Also, PARP is involved in the regulation of cellular signal transduction, transcriptional and methylation modification of dna, and cell cycle regulation. DNA damage activates a large amount of PARP to initiate repair function, and PARP inhibitors are effective in preventing DNA repair in cancer cells by inhibiting the function of PARP (158). However, most patients are resistant to PARP inhibitors, resulting in no significant improvement in the overall survival of patients treated with monotherapy (159). PAPR inhibitors promote the expression of PD-L1 to regulate the tumor immune microenvironment, leading to tumor cell resistance to the killing effect of toxic t cells. This also suggests that PD-1/PD-L1 inhibitors are capable of synergistic effects with them (160). The current study found that the use of a combination of PD-1/PD-L1 inhibitors and parp inhibitors in patients with triple-negative breast cancer and ovarian cancer inhibited tumor cell proliferation (161, 162). In a recent study, Wu et al. (163) conducted a retrospective analysis of 40 patients with solid tumors using a combination of PARP inhibitors and PD-L1 inhibitors. They analyzed the efficacy metrics [including overall survival (OS), disease control rate (DCR), progression-free survival (PFS) and objective remission rate (ORR)] and safety metrics (occurrence of grade 3 or greater adverse events) of nivolumab/pembrolizumab in combination with niraparib/olaparib and found that this combination efficacy metrics were better than with PD-1 inhibitors alone. In addition, the combination was found higher ORR in patients with gynecologic tumors and small cell lung cancer (164, 165). In 2018, the FDA approved Olaparib for use in metastatic breast cancer with mutations in the breast cancer susceptibility gene (BRCA) and human epidermal growth factor receptor 2 (HER2) negativity. By testing PD-1/PD-L1 inhibitors in combination with PARP inhibitors in a variety of cancers, the use of PARP inhibitors will become more widespread in the future.

Cancer is also considered to be a cell cycle disease (166) as cell cycle dysregulation is present in all human cancers and tumor cells often show genetic instability and abnormal proliferation. To date, scientists have conducted a series of studies on cell cycle regulation mechanisms, which have led to the emergence of anticancer drugs targeting the cell cycle as a hot topic of research. The CDK4/6-RB-P16 signaling pathway plays an important role in the regulation of the cell cycle. Abnormal proliferation of tumor cells resulting from abnormal regulation of this pathway is seen in various cancers (167). FDA has approved CDK4/6 inhibitors in HER2-negative, ER-positive postmenopausal breast cancer patients, which can block the binding site of cell cycle protein D1 on CDK4/6 thereby inhibiting tumor cell proliferation. Teh et al. (168) found that CDK4/6 inhibitors also could achieve anticancer effects by increasing the immunogenicity of tumors and downregulating immunosuppressive cells. However, CDK4/6 inhibitors alone are prone to drug resistance, and in order to be able to expand their use, researchers have conducted trials of various combination therapies. Goel et al. (169) found that the use of CDK4/6 inhibitors in patients with breast and colorectal cancer enhanced the sensitivity of tumor cells to anti-PD-1/PD-L1 antibodies through preclinical studies. However, further studies are needed to confirm the effectiveness of CDK4/6 inhibitors in combination with PD-1/PD-L1 inhibitors due to the lack of convincing trial data. To investigate whether CDK4/6 inhibitors and PD-1/PD-L1 inhibitors can act synergistically, Zhang et al. (56) constructed ovarian cancer mouse models and treated them with abemaciclib monotherapy and combined anti-PD-1 therapy, respectively. The results showed that the combination therapy provided better control of tumor cells relative to the single-agent group. In recent years, researchers have also found that CDK4/6 inhibitors improve the tumor immune microenvironment and enhance T-cell activity, and have been found to significantly increase the antitumor effect when combined with PD-1/PD-L1 inhibitors in a variety of animal models of cancer (168, 170).These studies also illustrate the potential of combining the two in the future treatment of cancer.

In addition to some of the drugs described above, the combination of molecularly targeted drugs such as BRAF inhibitors, MEK inhibitors, and adenosine receptor inhibitors with PD-1/PD-L1 inhibition has joined the research process of researchers. Mutations in the BRAF gene are closely associated with the development of melanoma, yet most melanoma patients are resistant to BRAF inhibitors (171). Frederick et al. (172) found this phenomenon to be associated with upregulation of PD-L1 expression. This also suggests the possibility that PD-1/PD-L1 inhibitors can increase the efficacy of BRAF inhibitors. Nevertheless, satisfactory results were not obtained in some clinical trials in melanoma patients using this combination (173, 174). When BRAF is mutated it activates the downstream MAPK pathway, leading to the activation of MEK, ERK and other proteins, which in turn regulate the processes of cell proliferation, growth and apoptosis (175). Researchers have found in clinical studies that the use of BRAF inhibitors in combination with MEK inhibitors in patients with metastatic melanoma can reduce the development of resistance to BRAF inhibitors in patients, thereby enhancing the therapeutic effect (176). Several investigators have used preclinical models to find that combining anti-PD-1 therapy with BRAF inhibitors and MEK inhibitors improves efficacy (177, 178). In a subsequent clinical trial, Ribas et al. (179) evaluated the safety and efficacy of this triple combination therapy and showed that it had a longer antitumor effect and PFS. although the probability of adverse events was also increased, this combination may be best suited for patients who do not respond well to monotherapy. The sequence and optimal duration of therapy for the combination of these three agents remains unclear, and their long-term efficacy requires continued attention, so longer follow-up and more studies are needed to assess the safety and efficacy of this combination. Adenosine is an immunomodulatory molecule that, when combined with adenosine 2A receptor (A2AR) and adenosine 2B receptor (A2BR), enhances the function of immunosuppressive cells (e.g., MDSCs and Tregs) and inhibits the activation of immunoprotective cells (e.g., dendritic cells, T cells, and NK cells), resulting in an immunosuppressive effect (180). Based on the ability of adenosine to inhibit T-cell function and the fact that upregulation of adenosine receptor expression levels can be found on activated T-lymphocytes after blockade of PD-1 has been shown (181), this also suggests that PD-1/PD-L1 inhibitors combined with adenosine receptor inhibitors have the potential to control tumorigenesis and progression. After Beavis et al. (182) evaluated this combination therapy in a mouse model of cancer, they found that this combination promoted IFNγ production by tumor-infiltrating CD8+ T lymphocytes. also they found that the tumor size in the combination group was significantly smaller than in the PD-1 monotherapy group, and that this potentiation was associated with IFNγ production. It is important to note that different dosing sequences of ICIs and small-molecule-targeted drugs may result in widely varying efficacy and adverse effects.