Tumor virus therapy induces immunogenic death on target cells and induces immune response by releasing pathogen-associated molecular pattern and damage-associated molecular pattern (15,16). As a result, tumor virus therapy improves the sensitivity of tumor cells to immunotherapy thus improving therapeutic effect. OVs mediates clearance of cancer cells or killing of cancer cells by targeting the tumor vascular system and inducing immunity (17). Talimogene laherparepvec, a herpes simplex virus expressing granulocyte-macrophage colony stimulating factor, was the first US FDA approved oncolytic therapy (18). Local intratumoral injection of the virus into tumors improves the overall survival rate of patients (19). A previous study (7) reported on the treatment of 21 patients with advanced melanoma with Talimogene laherparepvec combined with Pembrolizumab. The study report that therapy was well tolerated, with fatigue, fevers and chills as the common adverse events. The therapy showed no dose-dependent toxic reaction and an objective response rate (ORR) of 62%. Patients who responded to the combination therapy showed an increase number of CD8+T cells (7). Vaccinia virus is a highly immunogenic oncolytic immunotherapy vector (20,21). Previous studies report that vaccinia virus attracts effector T cells in mouse model of colorectal cancer and ovarian cancer (22,23). A combination of vaccinia virus with PD-L1 inhibitor enhances the infiltration of effector CD4+ and CD8+T cells and increases granzyme B, ICOS, perforin and IFN-γ, thus improving the survival rate (23).

PD-1/PD-L1 drug resistance is a main challenge, therefore, studies are required to explore novel approaches to improve immunogenicity of tumors and overcome resistance to immunotherapy (8). Rotavirus vaccine has immunostimulatory and anti-tumor effects (24). Administration of rotavirus in tumors overcomes drug resistance against PD-L1 inhibitors and has a synergistic effect with PD-L1 inhibitors. Heat- and UV-inactivated rotaviruses have no oncolytic activity but offer a synergistic effect with immune checkpoint-targeted antibodies through upregulation of the double-stranded RNA receptor retinoic acid-induced gene 1 (25). Rotaviruses have been used clinically and can be used for clinical sensitization of anti-PD-1/PD-L1 therapy (25) (Table I).

Tumor vaccine enhances immunogenicity and activates the immune system of the patient, thus controlling or eliminating tumors (26). DNA vaccine, a universal and personalized cancer treatment containing multiple new antigen coding sequences, is ideal for new antigen vaccination (27). DNA vaccine induces Cytotoxicity of CD8 T cells. A single dose of DNA vaccine combined with anti-PD-1 treatment significantly delays tumor growth in tumor-bearing mice inoculated with MC38 colon cancer cell line and some of the tumors are cleared completely, with a cure rate of 25%, and this indicates that tumor vaccine works synergistically with immune checkpoint blocking therapy (28).

Lmdd-MPFG vaccine promotes expression of PD-L1 in HCC cells but re-sensitizes tumor local T cell to respond to anti-PD-1 immunotherapy (29). Lmdd-MPFG vaccine activates NF-κB pathway and autophagy pathway in tumor-associated macrophages (TAMS). In addition, it converts M2 TAMS to M1 and induces the expression of antineoplastic factors, thus restoring T cell response to PD-1 inhibitors (29). Lmdd-MPFG vaccine acts synergistically with PD-1 inhibitors in treatment of liver cancer (29).

The tumor vaccine OVA @ Mn-DAP with nano-scale coordination polymer as a carrier, prepared from Mn²⁺ ions, Nod1 agonist and DAP as organic ligands, promotes maturation of dendritic cells and cross-presentation of antigens. Further, it prevents occurrence of B16-OVA tumors and works synergistically with PD-1 inhibitors to inhibit tumor growth (30).

Nivolumab combined with a multi-peptide vaccine (gp100, MART-1 and NY-ESO-1 with Montanide ISA 51 VG) was investigated as adjuvant therapy in resected stage IIIC and IV melanoma patients (31). The study findings showed that the treatment strategy was well tolerated. Common adverse events observed included fatigue, rash/pruritus, nausea/diarrhea, arthralgias and endocrinopathies. Although related grade 3 events occurred in 4 out of 33 patients, they were manageable. Notably, the combination therapy significantly increases CD8+ T-cell levels and decreases PD-1 expressing T-cells. In addition, significant increases in CD25+ regulatory T cells (Tregs)/CTLA4+/CD4+ T-cell populations are observed with anti-PD-1 therapy (31). These findings imply that synergistic activity of nivolumab and anti-PD-1 therapy is mediated through CTLA-4 and/or Tregs (32).

A previous phase I study (33) evaluated a vaccine that targets ≤20 predicted personal tumor neoantigens in patients with previously untreated high-risk melanoma following surgical resection. In that study, vaccine-induced polyfunctional CD4+ and CD8+ T cells targeted 58 (60%) and 15 (16%) of the 97 unique neoantigens used across patients, respectively. These T cells discriminated mutated antigens from wild-type antigens and recognized autologous tumors. Out of the six vaccinated patients, four showed no recurrence at 25 months following vaccination, whereas two showed recurrent disease and were subsequently treated with anti-PD-1 (pembrolizumab) therapy achieving complete tumor regression, with the expansion of a repertoire of neoantigen-specific T cells (33).

A previous study (34) explored the synergistic effect of a vaccine targeting HER2Δ16 on anti-PD-1 therapy in enhancing antitumor immunity in a model of advanced HER2⁺ breast cancer. HER2Δ16 is a critical oncogenic pathway and spontaneous tumors driven by HER2Δ16 are reflective of clinically advanced immunosuppressive HER2⁺ breast cancer. Endogenous HER2Δ16⁺ breast cancers show no response to anti-PD-1 as a single agent. Treatment with anti-PD-1 is not effective in increasing systemic anti-HER2 T-cell responses. However, combination of anti-PD-1 with Ad-HER2Δ16-KI significantly increases survival rate, with ~30% of mice exhibiting complete tumor regression and long-term tumor-free survival. These findings show that vaccinated mice are characterized by a high IFN-γ gene signature score. In addition, the results show that HER2Δ16 vaccination induces systemic adaptive immune responses and increases HER2-specific CD8 T cells that infiltrate into tumors. Therefore, addition of anti-PD-1 effectively induces HER2-specific T cells in TME (34) (Table II).

Chemotherapy usually kills cancer cells by targeting their DNA synthesis and replication (90). It also promotes the presentation of tumor antigens following cancer cell death, activates tumor specific T cells, facilitates DCs maturation, stimulates type I interferon response and eliminates bone marrow-derived immunosuppressive cells (91). Appropriate combination of chemotherapeutic drugs and PD-1/PD-L1 inhibitors can enhance the efficacy of PD-1 blockers and produce a more sustained anti-tumor response, especially in tumors with poor immunogenicity and sensitivity to chemotherapy. A study has shown that Pembrolizumab combined with pemetrexed/carboplatin enhances improves symptoms of metastatic non-squamous NSCLC and has been approved by US FDA (92). Application of Pembrolizumab in combination with pemetrexed and platinum increases the PFS of metastatic NSCLC (93). For untreated patients with metastatic squamous NSCLC, the PFS and OS of Pembrolizumab combined treatment group versus the placebo group were 6.4 months vs. 4.8 months and 15.9 months vs. 11.3 months, respectively. The risk of death decreased by 36% and the risk of disease progression or death reduced by 44% in the Pembrolizumab combined treatment group (94) (Table IV).

Radiotherapy (RT) has profound immunological effects. Basic research studies have demonstrated that RT can improve the efficacy of PD-1 inhibitors (95). Cancer cells can be killed by radiation. RT activates the immune system by triggering the release of tumor antigens (96). Basic research and clinical trials have revealed that RT synergizes with immunotherapy when applied together (97,98). A study has shown that PD-1 inhibitors combined with RT can activate CTLs and reduce immunosuppressive cells (99). A combination of RT and PD-1/PD-L1 inhibitors significantly improves the survival rate and reduces the tumor volume in mice (100). Compared with the control group, co-treatment of RT and PD-1 significantly increased the expression of PD-L1, CD8+T cells and interferon-γ in tumor cells (101). Clinical studies have provided evidence that anti-PD-1 therapy can significantly improve the control rate and OS rate of patients with melanoma brain metastasis who received stereotactic radiotherapy (102). Clinical trials of the efficacy of Nivolumab combined with RT in the treatment of NSCLC (NCT02768558) and glioblastoma (NCT02617589) are under way (103).

Immunotherapy amplifies the rare systemic effects of radiotherapy, while radiotherapy renders immune-excluded tumors quickly responsive to immunotherapy (104). MDSCs have been implicated in development of radioresistance. Accumulation of MDSC in the tumor microenvironment promotes tumor relapse by directly affecting tumor cell survival and indirectly affecting local T cell suppression (105). A combination of irradiation (IR) and anti-PD-L1 therapy enhanced the activation of CD8+ T cells and inhibition of TUBO tumor growth. CD8+ T cells induce the apoptosis of MDSCs through TNF-α following combination therapy (95). PD-L1 is upregulated in the tumor microenvironment following IR. IR-induced increases in tumor-infiltrating lymphocytes (TILs) and upregulation of PD-L1 could provide an opportunity for PD-L1 blockade (106). The combination of IR and anti-PD-L1 treatment optimizes the tumor immune microenvironment and results in tumor regression (95). Local radiotherapy significantly adds to the systemic efficacy of immunotherapy. Combining single-site stereotactic body radiotherapy (SBRT) with pembrolizumab improves response rates in metastatic NSCLC. PD-L1-negative patients benefited from SBRT (107). This study also suggested that the way to improve the effect of immunotherapy was to treat with local radiotherapy to synergize the local and systemic effects of both modalities. Immunotherapy increased the local effect of radiotherapy in all treated sites. Radiotherapy suppresses the tumor burden allowing immunotherapy better to eliminate micro-metastatic disease (108). In addition, a study demonstrated that higher single doses of RT from 12–18 Gy blunt the efficacy of anti-tumor immunity. They also reduce IFN-β production and abrogate DC-mediated CD8+T-cell priming, suggesting that RT doses below 12 Gy may be more immunogenic (109). Another study confirmed that a single dose of 15 Gy irradiation results in higher tumor immune cell infiltration than a fractionated (3 Gy × 5) schedule (110). The differences in the above results might be due to differences in the genetic backgrounds of mice, immune competence and immunogenicity of models and radiosensitivity of cell lines (111) (Table IV).

Intestinal microflora influences the effects of immunotherapy in cancers. A previous study reported that oral Bifidobacterium can significantly decrease the growth rate of melanoma, promote the maturation of dendritic cells and production of IFN-γ and enhance the anti-tumor effect of PD-1 inhibitors (112). The abnormal composition of intestinal flora may affect the response of patients to cancer immunotherapy (113). Transplantation of fecal bacteria improved the anti-tumor effect of PD-1 inhibitors (114). A study has shown that the clinical response of PD-1 inhibitors is dependent on the relative abundance of Akkermansia muciniphila. Oral supplementation of Akkermansia muciniphila restores the efficacy of PD-1 inhibitors in an IL-12-dependent manner (115). In another study, intestinal microflora regulated the response of anti-PD-1 immunotherapy to melanoma patients (116).

Patients with abundant beneficial intestinal bacteria (Ruminococcaceae/Faecalibacterium) have improved antigen presentation, effector T cells function in peripheral and tumor microenvironment and strong anti-tumor immune response (117). By contrast, the intestinal harmful bacteria (Bacteroidales) weakened antigen presentation and impaired anti-tumor immune response (118,119). Response to PD-1 inhibitors is influenced by the composition of intestinal flora, but not to oral flora (120). Other studies suggest that patients with melanoma responsive to Nivolumab were rich in Fecalibacterium prausnitzii, Bacteroides thetaiotamicron, B. longum, C. aerofaciens and E. faecium. Patients who responded well to Pembrolizumab were rich in intestinal Dorea formicogenerans (121,122) (Table V).

Diosgenin is a natural steroidal saponin (123). A combination of diosgenin with PD-1 inhibitor suppresses tumor growth, increased T cell infiltration and IFN-γ expression in tumor tissues. Diosgenin stimulates the immune cells thereby improving the response rate and therapeutic effect of PD-1 inhibitors (124). Diosgenin treatment downregulates intestinal Bacteroidetes but upregulated Clostridiales, Lactobacillus and Sutterella (124).

Icariin possesses a variety of pharmacological and biological activities. Icaritin is now under clinical trial for the treatment of PD-L1 positive advanced liver cancer (NCT03236649) and advanced breast cancer (NCT01278810). Pre-clinal studies have shown that Icaritin can effectively reduce the tumor load of B16F10 melanoma and MC38 colorectal cancer in mice and its therapeutic effect is T cell-dependent. It increased CD8 T cell infiltration and the number of effector memory T cells. A combination of PD-1 inhibitor and Icaritin significantly suppressed tumor growth (125).

Rhus verniciflua Stokes (RVS) has been shown to contain a large number of bioactive phytochemicals, including alkaloids, polyphenols and flavonoids, which block the interaction between PD-1/PD-L1 and CTLA-4/CD80. Thus, RVS might be used as an immune checkpoint blocker (126).

Ganoderma lucidum reduces the proportion of PD-1 positive cells in B lymphocytes. It can, therefore, be used to develop a new type of immunomodulator for the prevention and treatment of cancer (127). The combination of Ganoderma lucidum and paclitaxel inhibits the expression of immune checkpoints (PD-1 and Tim-3) and restored TILs. The combination regulates the development of 4T1-breast cancer in mice (128) (Table VI).

The anti-tumor response rate of PD-1 inhibitors is low. Patients sensitive to PD-1/PD-L1 inhibitors develop drug resistance, tumor recurrence and disease progression and the mortality rate of patients with advanced tumor stages is high. A study has reported that patients with melanoma sensitive to anti-PD-L1 antibody treatment show increased levels of interferon-γ and related genes in blood prior to treatment (129). Anti-PD-1 therapy downregulates expression of IFN receptor-related genes and MHC I and upregulates inhibitory receptors on the surface of T cells (10). Furthermore, the inhibitory receptors inhibit the cytotoxic activity of T cells and these effects can be attributed to drug resistance against ICIs. Several basic and clinical studies are exploring effective combination and sequence of PD-1/PD-L1 inhibitors and other anti-tumor therapies to induce tumor cell immunogenicity and improve effectiveness of anti-tumor effect of PD-1/PD-L1 inhibitors. These advances will provide effective therapies for patients who are unresponsive to current treatment regimens. However, development of these combination therapies possesses several challenges.

Development of effective antineoplastic therapy should consider medical costs and adverse reactions for each treatment. Therefore, it is necessary to determine predictive biomarkers for individualized therapy, so as to predict efficacy and adverse reactions of PD-1/PD-L1 inhibitors. At present, some patients are not sensitive to PD-1/PD-L1 inhibitors. Lack of biomarkers for predicting response rate limits the effectiveness of clinical treatment strategies, thus there is need to screen novel biomarkers for predicting immunotherapy responses in patients.

In order to increase the proportion of patients benefiting from PD-1/PD-L1 inhibitors, studies should explore potential predictive biomarkers for anti-tumor treatment. PD-L1 expression is a potential biomarker for predicting effectiveness of PD-1/PD-L1 immunotherapy on patients with cancer thus identifying patients who may benefit from immunotherapy. Expression of PD-L1 is associated with several TILs and activated tumor antigen-specific T cells induces expression of PD-L1 (130,131). However, expression of PD-L1 in tumor tissues is heterogeneous and changes with tumor treatment (132,133). Several staining antibodies are used in immunohistochemical methods (IHC) to detect PD-L1 expression and the staining techniques (manual and automated) vary (134–136). Currently, effectiveness of PD-L1 detection as an anti-tumor immune response index is still controversial. The association between the expression of PD-1 or PD-L1 at the tumor site and disease outcome varies in patients with different tumors (137–139). Therefore, it is difficult to achieve consistent results with PD-L1 detection, hindering application of anti-PD-1/PD-L1 therapy as precision medicine.

TIL in tumor tissue demonstrates the presence of immune response by the body (140). TIL positive + PD-L1 positive group show improved PD-1/PD-L1 inhibitor immune response compared with TIL negative + PD-L1 positive group. This implies that the number of TIL can predict efficacy of PD-1/PD-L1 inhibitors (141). TIL mainly infiltrates into tumor nests, tumor stroma and tumor invasive margins of tumor tissues and different parts have different associations with therapeutic effects (142). Therefore, it is necessary to further determine the association between the quantity and quality of TIL and other infiltrating immune cells and tumor immune response. In addition, local radiotherapy is effective in inducing inflammation, which may benefit patients without sustained immune response (99). Radiotherapy should not be used in patients with significant tumor infiltration, as this may impair the ongoing immune response (143,144). Effects of different therapies on immune response should be considered when designing combination therapies with chemotherapy and targeted therapy.

Mismatch repair (MMR) is a set of susceptibility genes isolated from hereditary non-polyposis colorectal cancer. Mutations in these gene leads to loss of mismatch repair function, resulting in microsatellite instability (MSI) which is prone to tumors (145). Microsatellite instability high (MSI-H) attracts tumor-infiltrating lymphocytes (TILs) and upregulates PD-L1 expression in tumor epithelial cells (146). MMR deficiency (MMR-D) type solid tumors have more tumor neoantigens to enhance anti-tumor immune response and show an improved response to PD-1 monoclonal antibody, thereby improving immune suppression and restoring anti-tumor immunity (147). MMR-D is a predictor for anti-PD-D efficacy. However, MMR-D only occurs in a small number of patients. Further pre-clinical and clinical research should be performed before clinical application.

The therapeutic effect of PD-1 inhibitors is high in patients with a high mutation load of tumor mutation burden (TMB). Tumor cells with high TMB expression have higher levels of neoantigens, which stimulate a stronger anti-tumor immune response (148). TMB and PD-L1 have similar predictive function. However, TMB is not associated with PD-L1 expression. TMB is an important and independent predictive biomarker, which can predict the effectiveness of ICIs (149).

Further studies should explore ways to alleviate side effects of immunotherapy. Resistance of malignant tumors against PD-1/PD-L1 inhibitors can be overcome by use of combination therapy of PD-1/PD-L1 inhibitors (150). Notably, a combination of anti-PD-1/PD-L1 therapy is more effective compared with use of anti-PD-1/PD-L1 inhibitors alone. However, combination therapy is associated increased side effects (88). A study revealed that patients younger than 65 years old benefit more from nivolumab plus ipilimumab treatment than patients older than 65 years old. Therefore, combination therapies with ICIs should be carefully chosen for patients >65 years of age (151). Goals for treatment of patients with advanced cancer is usually palliative, prolonging survival, controlling symptoms and improving quality of life. Therefore, studies should explore combination therapies with ICIs and fully understand the toxic effects of immunotherapy, chemotherapy and radiotherapy to make sound treatment decision. Side effects such as immune disorders caused by ICIs are called immune-related adverse events. Common adverse reactions include diarrhea, fatigue, itching, rash, nausea and loss of appetite. Severe adverse reactions include severe diarrhea, colitis, myocarditis and cardiac insufficiency, liver dysfunction, pneumonia and glomerulonephritis (88,152,153). Serious side effects may require discontinuation of treatment, although patients may have an immune response thereafter. Intravenous corticosteroids or immunosuppressive drugs should be given if necessary. Some treatment-related autoimmune responses, such as rashes, are associated with improved prognosis (154). This implies that occurrence of adverse reactions is manifested by activation of immune system and represents action of PD-1/PD-L1 inhibitor, which eliminates tumors. There is an overlap between autoimmune reaction and anti-tumor immune reaction. Further studies should be performed to explore adverse drug reactions associated with immunotherapy.

Clinical application of molecular targeted drugs is associated with challenges such as acquired drug resistance and side effects which need to be minimized. Several studies are exploring the development of molecular targeted drugs with higher efficiency and fewer side effects (155,156). Studies exploring sequence, dosage and safety of PD-1 inhibitors and EGFR should be performed (157,158). Development of combination therapies will improve efficacy and reduce side effects of molecular targeted drugs. A number of clinical studies on combination therapies between chemotherapy and radiotherapy with immunotherapy are underway. An open-label, randomized phase 3 study showed that pembrolizumab+chemotherapy significantly improved OS in the total population. The data support the use of pembrolizumab+platinum +5-FU as new first-line standards of care for recurrent/metastatic head and neck squamous cell carcinoma (NCT0235803) (159). In the KEYNOTE-189 and KEYNOTE-407 studies (phase III), PFS and OS were significantly longer in patients treated with pembrolizumab and chemotherapy compared with those in patients treated with chemotherapy alone (93,94). Anti-PD-1 therapy enhances the efficacy of radiotherapy in metastatic gastric cancer treatment by increasing the CD8⁺ T cell/effector regulatory T cell ratio in TILs (160). Another study showed that patients with metastatic NSCLC treated with nivolumab or pembrolizumab+radiotherapy did not have increased grade 3/4 immune-related adverse events (161). The combination of chemotherapy and radiotherapy with PD-1/PD-L1 inhibitors induces lasting immune response in treatment of tumors when other treatment strategies fail.

Despite a significant number of basic and ongoing clinical trials aimed at improving effectiveness of combination therapies, intestinal flora combined with PD-1/PD-L1 inhibitors is a novel approach for cancer treatment. However, differences between basic and clinical trial results occur due to high variability of bacteria in intestinal tract and the effects produced by bacteria in the laboratory. Less diverse bacteria used in basic trials may not fully represent the complicated environment in the intestinal tract. Therefore, further studies should explore the mechanism of intestinal flora, side effects, optimal dosage and species for human use for development of effective combination therapies.