Chemoimmunotherapies are attractive, not only because they directly lead to the apoptosis of tumor cells and induce the release of antigens that can drive immune responses, but also because they suppress essential immune regulatory mechanisms that prevent the development of immunity [52]. One previous study revealed that chemotherapeutic agents including paclitaxel, etoposide, and 5-fluorouracil can induce or increase PD-L1 expression on human breast cancer cell lines [53]. Both paclitaxel and etoposide induced dose-dependent increases in PD-L1 surface expression in the human breast cancer cell line MDA-MB-468, whereas 5-fluorouracil was able to induce PD-L1 surface expression in breast cancer cell lines MDA-MB-468 and MDA-MB-435. Induced or increased PD-L1 expression has been considered the prerequisite for the clinical effectiveness of anti-PD-1 immunotherapy.

Currently, platinum-taxane-based adjuvant chemotherapy is the first-line treatment for OC after surgery. Cisplatin promotes its cytotoxicity by forming DNA-protein cross-links, DNA monoadducts, and intrastrand DNA cross-links, all of which trigger cell apoptosis [54]. Cell death can be classified according to the morphological change induced by the lethal process (apoptotic, necrotic, autophagic, or related to mitosis), enzymological criteria (the involvement of nucleases or different types of proteases), functional aspects (programmed or accidental and physiological or pathological), and immunological characteristics (immunogenic or nonimmunogenic) [55]. Although cisplatin does not induce immunogenic tumor cell death, Qin et al. [56] demonstrated that cisplatin can upregulate PD-L1 expression on hepatoma H22 cells; thus, PD-1/PD-L1 expression may represent a promising target for immunotherapy. Unlike cisplatin, oxaliplatin can lead to immunogenic tumor cell death, which is characterized by the exposure of chaperones—including calreticulin, shock proteins, or both—on the cell surface. The cell surface molecules then determine the uptake of tumor antigens and affect the maturation of DCs [55]. Paclitaxel, a member of the taxane family, not only selectively arrests the cell cycle in the M phase, but also inhibits the function of the apoptosis inhibitor protein BCL-2 [57]. In one study of stage II/III breast cancer, increased T cell blastogenesis and NK cell cytotoxicity was found in patients who received taxane treatment compared with patients who did not receive taxane treatment [58]. Moreover, in an OC mouse model, paclitaxel increased the number of CD8⁺ T cells in the tumor site, upregulated PD-L1 expression, and activated nuclear factor-κB signaling. Tumor-bearing mice treated with a combination of paclitaxel and PD-L1/PD-1 signal blockade were discovered to survive longer than mice treated with paclitaxel alone [20].

BRCA1 and BRCA2 genes are important components of the homologous recombination pathway. Approximately 17% and 6% of patients with high-grade serous carcinoma (HGSC) have been estimated to exhibit germline and somatic mutations in these genes, respectively [59]. Poly (adenosine diphosphate [ADP]-ribose) polymerase (PARP) plays a significant role in single-stranded DNA break repair and genomic stability through the base excision repair pathway [60]. PARP inhibition causes the death of BRCA-mutated cells through the synthetic lethality mechanism. Olaparib (Lynparza™; AstraZeneca Pharmaceuticals LP, Wilmington, DE, USA) is a PARP inhibitor that was approved by the FDA in December 2014 for the treatment of patients with germline mutations in BRCA1/2 [61]. One previous study showed that BRCA1- and BRCA2-mutated OC tumors exhibit increased CD3⁺ and CD8⁺ TILs and increased PD-1/PD-L1 expression on tumor-associated immune cells [21]. Mechanistically, PARP inhibitors inactivate glycogen synthase kinase-3β, which in turn improves PARP-inhibitor-mediated PD-L1 upregulation. Accordingly, PD-L1 pathway blockade resensitizes PARP-inhibitor-treated tumor cells to T cell killing. The combined use of a PARP inhibitor and anti-PD-L1 immunotherapy can significantly increase therapeutic efficacy for breast cancer compared with each agent alone [62]. Therefore, BRCA1- and BRCA2-mutated OC patients may be a suitable candidate population for these inhibitors.

Bevacizumab is a humanized mAb that inhibits angiogenesis by blocking the binding of vascular endothelial growth factor (VEGF)-A to VEGF receptor 1 (VEGFR1) and VEGFR2. It has been demonstrated that VEGF/VEGFR signaling can result in dysfunctional vasculature that inhibits the infiltration of T cells into the tumor site and reduces DC differentiation and activation, which may impair T cell priming. In addition to its activity on tumor vasculature for modulating host immunity, VEGF-A exerts direct inhibitory effects on immune cells, such as increases in the expression of checkpoint molecules on CD8⁺ T cells and the modulation of proliferation of Tregs. Treatment of mouse models of cancers such as colorectal cancer, lung cancer, cervical cancer, and OC with inhibitors of VEGF-A/VEGFR signaling increases T cell recruitment and infiltration into the tumor site, and it may be synergistic with anti-PD-1 therapy [63]. One study found that simultaneous treatment with anti-PD-1 and anti-VEGFR2 mAbs inhibited tumor growth synergistically in a mice model of colon-26 adenocarcinoma without overt toxicity [64]. An ongoing phase-II study is evaluating the safety and efficacy of PD-L1 in combination with bevacizumab in recurrent platinum-resistant OC. VEGF/VEGFR signaling pathways and the approved therapeutic agents targeting these pathways are illustrated in Fig. 3.

Epidermal growth factor receptor (EGFR) is a tyrosine kinase receptor, and the binding of EGF to EGFR induces the tyrosine phosphorylation of diverse cellular proteins. Increased EGFR expression in tumors indicates that the tumors are more aggressive, more likely to metastasize, and more resistant to chemotherapy, which lead to poorer outcomes in cancer patients. Cetuximab (Erbitux^®; Merck & Co., Inc.) binds to the extracellular domain of EGFR, inhibiting EGFR signaling and accelerating receptor internalization [65]. Treatment of OC cell lines in vitro with cetuximab suppresses cell growth, potentiates cell apoptosis, and impairs tumor metastasis [66]. Multiple trials are investigating strategies for the combined application of cetuximab with anti-PD-1/PD-L1 agents in various cancers, such as recurrent or metastatic squamous cell cancer of the head and neck (NCT02358031) and metastatic colorectal cancer (NCT02934529).

According to the dualistic model of carcinogenesis, epithelial OC can be categorized as type I and type II. Type I cancers consist of low-grade serous, low-grade endometrioid, clear cell, and mucinous carcinomas, as well as Brenner tumors. They are relatively indolent, present in early stages, and include specific mutations (KRAS, BRAF, ERBB2, CTNNB1, PTEN, PIK3CA, ARID1A, and PPPR1A) targeting specific cell signaling pathways. Type II cancers consist of HGSC, high-grade endometrioid, malignant mixed mesodermal tumors, and undifferentiated carcinomas. They are relatively aggressive, present in advanced stages, and harbor a high frequency of TP53 mutations [67]. Next generation sequencing (NGS) technology can be used for whole-exome and whole-genome sequencing. Studies have demonstrated that patients with a high frequency of somatic mutations are more likely to benefit from treatment with PD-1 inhibitors. The enhanced mutation load may activate adaptive immunity and attract CD8⁺ cell infiltrates. Thus, genomic analysis of the total mutational load using NGS can be employed to determine the population that will benefit from combined immunotherapy [68].

Ionizing irradiation is one of the most common treatment strategies for cancer. Radiation predominantly induces DNA damage in tumor cells through base damage, base release, depolymerization, crosslinking, and strand breakage, consequently leading to the apoptosis, necrosis, mitotic catastrophe, autophagy, or senescence of the cells [2269]. Following radiotherapy, cancer cells release various substances such as IL-6, IL-8, and tumor necrosis factor (TNF)-α, which can stimulate the immune system [22]. Zeng et al. [70] found that anti-PD-1 immunotherapy combined with stereotactic radiotherapy significantly prolonged the survival of glioma-tumor-bearing mice and generated long-term antitumor memory. Testing of long-term antitumor memory revealed that when naïve and “cured” mice (animals surviving >90 days after intracranial tumor implantation in combined immunotherapy group) were rechallenged using flank injections of GL261-luc cells, none of the “cured” mice had developed tumors by day 60 after implantation whereas 100% (8/8) of the naïve mice had developed flank tumors of size >1,000 mm³ by day 20 after implantation. The release of diverse tumor-associated antigens in a proinflammatory environment has been speculated to act as a vaccine, leading to the generation of immunologic memory. In melanoma, colorectal, or breast cancer cell lines, low doses of fractionated radiotherapy were demonstrated to lead to PD-L1 upregulation on tumor cells. Notably, fractionated radiotherapy combined with PD-1 or PD-L1 mAbs produced efficacious CD8⁺ T cell immune responses that improved long-term survival and protected against tumor rechallenge [71]. In OC cell lines, high doses of gamma irradiation (5,000–10,000 cGy) were confirmed to induce a significant and long-lasting upregulation of MHC class I (MHC I), MHC II, and antigens (CA125 and Her2-neu) expressed on the OC cell lines. The enhancement of antigen expression, which was crucial for both the recognition and destruction of OC cells by the host immune system, was persistent until all cells had died [72]. Deng et al. [73] reported that radiotherapy combined with anti-PD-L1 immunotherapy reduced the number of MDSCs, which is characterized by the surface makers of CD11b⁺ and Gr-1⁺, thus reducing the suppressive effects on the immune system. Therefore, the combination of immunotherapy with radiotherapy and PD-1 signaling blockade may be an effective antitumor strategy for improving treatment outcomes for cancers including OC.

CTLA-4 (also known as CD152) was identified in 1987 as the first coinhibitory molecule that plays a significant role in the down-regulation of T cell activity in vivo to limit self-damage [74]. CTLA-4 has a high affinity for CD86/CD80 and can thus effectively outcompete CD28 (a costimulatory molecule) for its ligands and deliver inhibitory signals to T cells [75] (Fig. 2). The precise mechanism of CTLA-4-mediated suppressive signaling is currently being explored. CTLA-4 has been postulated to suppress T cell function by inducing the dephosphorylation of the ζ chain of the TCR complex, through the Lck-dependent recruitment of SHP-2 [76]. Another study reported that CTLA-4 also suppresses T cell function by inhibiting signals from CD3-ε and in vitro evidence suggested that TCR-ζ and CD3-ε chains exert different effects as a result of heterogeneity in the primary sequence of their immune receptor tyrosine-based motifs [77]. TCR chains are physically associated with a set of invariant molecules: CD3-γ, CD3-δ, CD3-ε, and the TCR-ζ and η chains. The TCR-ζ chain plays a vital role in linking TCR-triggering to several protein tyrosine kinases—such as ZAP-70, Syk, Lck, and Fyn—which in turn control signal transduction pathways that regulate lymphokine and lymphokine receptor gene expression [78]. CD3-ε is critical in T cell thymic differentiation and functional competence [7980]. The administration of therapeutic mAbs to block CTLA-4 can prevent immune inactivation through the reversion of the aforementioned mechanisms and induce rational antitumor immune responses through the inhibition of Treg cell function and enhancement of effector CD4⁺ T cell activity [81]. Moreover, CTLA-4 and the PD-1 pathway are believed to operate during different stages of an immune response. CTLA-4 regulates T cell proliferation early and mainly in lymph nodes, whereas the PD-1 pathway suppresses T cell proliferation later and primarily in peripheral tissues [82]. These key similarities (both can induce immunosuppression) and differences (stages of immune response) of CTLA-4 and the PD-1 pathway indicate the possibility of their combination for cancer therapy. To date, 2 CTLA-4-targeting agents (tremelimumab and ipilimumab) have been assessed in clinical trials [83].

Tremelimumab is a fully humanized immunoglobulin G2 (IgG2) mAb that stimulates the proliferation of CD4⁺ T cells, IL-2 production by CD4⁺ T cells, and the proliferation of CD8⁺ cells [8485]. Tremelimumab blocks the binding of CD80/CD86 to CTLA-4, inhibits CTLA-4-mediated down-regulation of T cell activation, and enhances anticancer immunity through the potential aforementioned mechanism. Ipilimumab is a fully humanized IgG1-kappa recombinant mAb that inhibits CTLA-4; it was approved by the FDA in 2011 for clinical use in metastatic melanoma patients [83].

PD-1 and CTLA-4 are inhibitory cell surface molecules, and their blockade has been found to have promising efficiency in various solid tumors such as colon carcinoma and OC. Blockade of both PD-1 and CTLA-4 was previously shown to lead to tumor rejection in 2/3 of mice [85]. The double blockade was associated with the increased proliferation of antigen-specific effector CD8⁺ and CD4⁺ T cells, antigen-specific cytokine release (granzyme B, interferon (IFN)-γ, TNF-α), and the inhibition of the suppressive functions of Tregs. A previous study demonstrated that in the presence of anti-CTLA-4 mAb, spleen cells exhibited improved production of IL-2 and IFN-γ in mice with early fibrosarcoma and OC. Moreover, the mixed lymphocyte reaction (MLR) calculated from the uptake of ³H thymidine showed that the level of T cell proliferation increased with the dose of anti-CTLA-4 mAb. The administration of anti-CTLA-4 mAb at a high dose (20 µg/mL) markedly enhanced MLR [86]. Because preclinical data has demonstrated the synergistic efficacy of a combined PD-1/CTLA-4 blockade, the focus of research has shifted to the combined immunotherapy of the PD-1/CTLA-4 blockade [87]. Several clinical trials investigating the anti-PD-1/anti-PD-L1 antibody and anti-CTLA-4 antibody have identified marked clinical effects in numerous solid tumors [88]. In patients with melanoma, a total of 53 patients received concurrent therapy with nivolumab (anti-PD-1 mAb) and ipilimumab (anti-CTLA-4 mAb). The objective response rate in the concurrent-regimen group was 40%, with a manageable safety profile. The combined immunotherapy demonstrated more rapid and deeper tumor regression in a substantial proportion of patients than has been reported in published data on monotherapy [88]. Based on preclinical studies on OC and clinical trials of other malignancies treated using combined immunotherapy, there is an ongoing phase-II study using nivolumab both alone and in combination with ipilimumab in recurrent epithelial OC that aims to identify the objective tumor response rate (NCT02498600) [89]. Other ongoing clinical trials are listed in Table 2.

In addition to the aforementioned active agents investigated in clinical trials in OC patients, several new combined applications have been tested in animal models. OX40 (also known as CD134) is a costimulatory molecule belonging to the TNF receptor family and is primarily expressed on activated T cells—including CD4 T, CD8 T, type 1 T helper (Th1), Th2, and Th17 cells—and Tregs. However, naïve CD4, CD8, and most memory T cells do not express OX40 [24]. One study confirmed that the combination of antagonistic anti-PD-1 antibodies with agonistic anti-OX40 antibodies markedly inhibited tumor outgrowth in an OC model by increasing CD4⁺ and CD8⁺ T cells and decreasing immunosuppressive CD4⁺ FoxP3⁺ Treg cells and CD11b⁺ Gr-1⁺ MDSCs [90]. Wei et al. [91] discovered that the combination of anti-PD-1 mAbs and anti-CD137 mAbs doubled the OS. Mice with OC that were treated using this combination exhibited a significantly increased total number of CD8⁺ T cells, both in peritoneal lavage and spleens, which were determined to be functional using their antigen-specific cytolytic activity and IFN-γ production. Moreover, adding cisplatin to the combination of the 2 mAbs increased OS by more than 90 days. Lu et al. [92] discovered that the costimulatory receptor glucocorticoid-induced TNFR-related protein (GITR) is upregulated on activated CD4⁺ and CD8⁺ T cells; GITR engagement increases the proliferation and activation of these T cells and the production of cytokines such as IFN-γ by these T cells. Concomitant PD-1 blockade and GITR triggering can remarkably inhibit ID8 ovarian tumor growth and synergistically improve OS in mice. The durable antitumor effect was associated with the increased frequency of IFN-γ-producing effector T cells and decreased immunosuppressive Tregs and MDSCs, which changed an immunosuppressive milieu to an immunostimulatory state in the peritoneal cavity. In addition, combined treatment induced an antigen-specific immune response, as evidenced by antigen-specific IFN-γ production and the cytolytic activity of spleen cells in treated mice. Moreover, treatment with anti-PD-1/GITR mAb combined with chemotherapeutic drugs (cisplatin or paclitaxel) further increases the antitumor efficacy. Although the detailed mechanisms of synergy between combined immunotherapy and cisplatin/paclitaxel remain unclear, it is speculated that their synergy is partially due to increased tumor antigenicity and the chemotherapy-induced death of immunosuppressive cells. Trabectedin (ET-743, Yondelis^®; Janssen Products, LP, Beerse, Belgium) is a synthetic, marine-derived anticancer agent that can arrest the cell cycle and is currently used to treat patients with relapsed OC. Trabectedin binds to the minor groove of duplex DNA, bends the DNA helix to the major groove, and causes perturbation of the cell cycle [93]. Tumor cells in the G1 phase are more susceptible to being delayed by trabectedin than tumor cells in the S and G2+M phases [9495]. Treatment of mice with OC using combined α-PD-1 mAb and trabectedin induced a strong antitumor immune response, resulting in notable tumor inhibition; half of these mice were tumor-free for 90 days after OC cell inoculation. A mechanistic investigation revealed that the combination treatment remarkably increased the percentage of effector CD4⁺ and CD8⁺ T cells and concomitantly decreased that of immunosuppressive Tregs and MDSCs, resulting in a substantial elevation of the ratios of effector CD4⁺ and CD8⁺ T cells to immunosuppressive Tregs and MDSCs. Moreover, the combined α-PD-1 mAb and trabectedin treatment was also found to alter the immune microenvironment by increasing Th1 effector T cell recruitment and functionality (i.e., increased transcripts for IFN-γ, IL-12p40, and T-bet) [96].