Research Progress of Immuno-Inhibitory Receptors in Gynecological Cervical Cancer

Abstract

The mortality rate of cervical cancer is the highest among female malignant tumors and seriously threatens women's lives and health. Persistent high-risk human papillomavirus (HPV) infection is the leading cause of cervical cancer, which provides the basis for immunotherapy. In recent years, owing to progress in targeted therapy and immunotherapy, the survival time of patients with cervical cancer has been significantly extended. However, effective treatments for advanced, recurrent, and metastatic cancers are lacking. “Tumor immunotherapy” has been described as a viable option for tumor therapy but the efficacy of immunotherapy for cervical cancer has only been demonstrated in phase I or II clinical trials. Immune checkpoint inhibitors (ICIs) have shown promising clinical results particularly for treating recurrent and advanced cervical cancer, however, they remain inadequate in some patients. Immune checkpoint is the target of immunotherapy. Therefore, the identification of novel therapeutic targets is essential. In this paper, the structure, expression, function, biological effect of immune inhibitory receptors (IRs) and related clinical studies were reviewed, in order to further explore the application potential of these immune checkpoints and apply them to the future clinical treatment of cervical cancer.

Introduction

Epidemiology of Cervical Cancer

Cervical cancer is the fourth most common cancer in women worldwide. In 2020, there were 604,127 new cases of cervical cancer and 341,831 related deaths, ranking fourth in both the incidence and mortality of malignant tumors in women. The most important risk factor for cervical cancer is high-risk HPV infections (mainly HPV16 and HPV18). However, other factors can also contribute to the development of cervical cancer, starting sex too early, having too many sexual partners, not using condoms, multiple pregnancies, and infection with pathogens such as chlamydia and HIV. ¹ Due to the gap in cervical cancer screening, HPV infection prevention, and control levels, the mortality rate of cervical cancer has significant regional differences. Cervical cancer can be prevented and cured if detected early and treated appropriately. However, in developing countries, owing to a lack of planned HPV vaccination, early physical screening and appropriate sex education, the incidence of this cancer is high and it is only detected at an advanced stage. In low-income areas such as East Africa, the mortality rate of cervical cancer can be as high as 30.0/100,000, whereas, in high-income regions such as Australia and New Zealand, the mortality rate is only 1.7/100,000, a gap of 17 times. Advanced cervical cancer is a significant cause of death in women. For example, the 5-year survival rate for patients with metastatic cervical cancer is 16.5%, while the 5-year survival rate for patients with localized cervical cancer is 91.5%. Early treatment options include surgery and radiotherapy, and later radiotherapy combined with cisplatin-based chemotherapy. However, these treatments are only effective in patients with early-stage disease and limited metastasis. In addition, a pivotal point to consider is the recurrence rate in patients with cervical cancer, which is low in patients without metastasis and high in those with metastasis.^2,3 Therefore, high recurrence and mortality rates are associated with immune system escape, resistance, invasion, and metastasis. These issues necessitate the development of targeted immunotherapies, which have increased in recent years. However, treatment options for advanced cervical cancer remain limited.

The Immune Pathogenesis of Cervical Cancer is Related to IRs

At present, immunotherapy is the most prospective oncology treatment. Immune checkpoint is the target of immunotherapy, it is the receptor of immune cells or tumor cells, and plays a positive or negative role in the regulation of the immune system. Immune checkpoints can be divided into two groups according to their role in the immune microenvironment: immune activation checkpoints and immunosuppressive checkpoints. The expression of inhibitory receptors (IRs) is involved in the negative regulation of immune response, maintaining tolerance and preventing autoimmunity under physiological conditions. However, in the tumor microenvironment, dysregulation of immune checkpoint receptors evades the anti-tumor immune response. ⁴ The immunopathogenesis of cancer is distinct, and CD8 + T cells are mainly responsible for tumor recognition and antigen-specific functions. After recognizing cancer cells, tumor antigen-specific T cells can secrete interferon-γ (IFN-γ) and other cytokines, inhibiting the function of CD8 + T cells and thus inhibiting the elimination of cancer. ⁵ Tumor-specific T cells must target highly immunogenic tumor antigens. Specific T cell target antigens are abundant in various cancers presenting with DNA mutations, such as ultraviolet radiation-induced malignant melanoma, smoking-related lung cancer, DNA repair gene abnormalities such as BRCA1/2 associated tumors, and tumors with high microsatellite instability (MSI). In addition, long-term HPV infection is closely associated with the carcinogenic effects of cervical cancer; therefore, cervical cancer is also considered a T-cell target antigen-associated tumor. Cervical cancer has particular characteristics of the tumor genome and microenvironment, predicting that combination immunotherapy has a high probability of response. First, a majority of cervical cancers have a high tumor mutation load and a T-cell inflammatory gene signature known as immune fever. There is a sustained immune response in immune-thermal tumors, but this response is suppressed by immune escape or immunosuppressive pathways in the tumor microenvironment.^6,7

The immune cell activation response to HPV infection begins with keratinocytes, which are a part of the innate immune system. In addition, keratinocytes can express different pattern recognition receptors, such as toll-like receptors (TLRs), and produce cytokines, such as IFN-γ, TNF-α, IL-18, and specific chemokines. These cytokines and chemokines promote the recruitment and activation of immune system cells, such as NK, CD4+, and CD8 + cells, macrophages, dendritic cells, and other cells that are important in activating the immune system.^8–10

In 1983, German scientist Dürst ¹¹ first hypothesized that the occurrence of cervical cancer may be related to HPV infection and has been clinically verified, which was of landmark significance for the etiological research of cervical cancer. With advances in tumor cell and molecular biology, HPV genotypes have been isolated and identified. Over 150 HPV subtypes have been identified, of which 12 are carcinogenic. For high-risk HPV types, such as 16 and 18, continuous infection can lead to integration with the host genome, resulting in the overexpression of oncoproteins E6 and E7. ¹² The carcinogenicity of HPV is closely related to the viral proteins E6 and E7. E6 and E7 have no enzymatic activity and regulate cellular biological activities by interacting with host intracellular proteins to form complexes. These oncoproteins down-regulate interferon production, interfere with natural immune responses via the STING pathway, and inhibit HPV antigen expression via Class I MHC molecules. In addition, HPV infection can impair the function of Langerhans cells, which play a crucial role in the immune response.

The mechanism of carcinogenesis differs among virus-induced cancers, resulting in different mutation loads in the tumors. Carcinogenic viruses destroy the host cell structure, causing viral DNA to bind to host cytokines and induce a DNA damage response (DDR). The DDR increases the mutation rate and accelerates changes in host chromosomes, thus promoting viral replication. HPV is a classic example of promoting DDR and activating mutations. ¹³ It has been reported that the conformational change of the E6 protein after binding with the cellular ubiquitin ligase E6AP can specifically bind p53 to form the E6/E6AP/p53 complex, thereby enabling the ubiquitination and degradation of p53. In addition, the E7 protein also binds to and inactivates p-Rb. The inactivation of the tumor suppressor genes p53 and p-Rb blocks cell cycle progression and apoptosis, resulting in excessive cell proliferation and carcinogenesis. ¹⁴ Studies have shown that when transfected into prostate cancer cell lines, HPV E7 increases the expression of programmed cell death protein-1 (PD-1). When E7 was knocked down in CaSki cells, the expression of Programmed death ligand-1 (PD-L1) was reduced. Subsequently, T cell proliferation and cytotoxic activity increase. ¹⁵ Immune system plays a vital role in HPV-induced carcinogenesis, and the interaction between the tumor immune microenvironment and HPV host cells determines the progression of cervical cancer. ¹⁶ The relationship between tumors and the immune system can be divided into three stages. In the clearance stage, the newborn tumor has strong immunogenicity and can be identified and cleared by the innate and acquired immune systems. A few surviving tumor cells exhibit weakened antigenicity and evade immunity. The clearance function of the system enters the second stage, the equilibrium stage, in which tumor cells are still under the clearance pressure of the immune system and cannot overgrow. When a tumor gene mutation is involved to a certain extent, this balance is disrupted; that is, it enters the escape stage and produces a series of malignant phenotypes. ¹⁷

In the healthy state, when antigen-specific T cells are activated, the expression of checkpoint receptors is upregulated to reduce pro-inflammatory responses. However, with long-term activation of persistent antigens, such as chronic viral infections or cancer, checkpoint expression is maintained, and effector T cells enter a “depleted” state. The proliferative capacity of T cells is gradually reduced, and effector T cell functions, including the production of inflammatory cytokines and degranulation, are lost. Consequently, therapies targeting these checkpoint receptors have been developed to restore the effector functions of depleted T cells. Normally, dendritic cells engulf cancer cells, migrate to lymph nodes, and present them to T cells in the lymph nodes. T cells recognize cancer cells; however, the immune signaling of T cell functions needs to be determined.^17,18 These signals can either be mutually reinforced or inhibitory. In the promotive form, T cells are activated, inversely, in the inhibitory form, T cells recognize but cannot attack the cancer cells. Additionally, in the tumor microenvironment, inhibitory signals are expressed on the surface of tumor cells to inhibit the immune function of activated T cells in lymph nodes. Therefore, the lymph node and tumor microenvironment have different mechanisms for determining the direction of the immune response against cancer cells. These signaling molecules are called immune checkpoints. ⁴ IRs play an inhibitory role in immune checkpoints in the immune system, and the most common inhibitory receptors are PD-1, Cytotoxic T-lymphocyte antigen 4 (CTLA-4), T cell immunoglobulin domain and mucin domain-3 (TIM-3), T cell immunoreceptor with Ig and ITIM domains (TIGIT) and Lymphocyte activationgene-3 (LAG-3) (Table 1). Typically, these checkpoints regulate T-cell function to maintain autoimmune tolerance and prevent an autoimmune response. However, in tumorigenesis, they become “accomplices” for tumor immune escape. ⁴

Table 1.

Comparison of Different Inhibitory Receptors (IRs). ⁴

Receptor	Synonyms	Ligands	Expressing cells	Ligand-expressing cells	Function
PD-1	CD279	PDL-1 and PDL-2	Activated T cells, Treg, NK, B cells, macrophages, exhausted effector T cells	APCs, tumor cells, hematopoietic cells	Inhibition
CTLA-4	CD152	CD80/CD86	Activated T cells, Tregs, exhausted effector T cells	APCs	Inhibition
TIM-3	HAvcR2	Galectin-9	Activated T cells, DCs, NK, B cells, macrocytes, exhausted effector T cells	APCs and tumor cells	Inhibition
TIGIT	WUCAM	CD155(PVR) and CD112	Activated T cells, Tregs, NK, exhausted effector T cells	tumor cells and tumor-associated myeloid cells	Inhibition
LAG-3	CD223	MHC II class, LSEC tin, Galectin-3, FGL-1	Activated T cells, Tregs, NK, NKT, activated B cells, natural plasma cells, exhausted effector T cells	APCs, hepatocytes, monocytes, tumor cells	Inhibition

PD-1: Programmed cell death protein-1, PD-L1/2: Programmed death ligand-1/2,CTLA-4:Cytotoxic T-lymphocyte antigen 4,TIM-3:T cell immunoglobulin domain and mucin domain-3,TIGIT: T cell immunoreceptor with Ig and ITIM domains, PVR: poliovirus receptor, LAG-3:Lymphocyte activationgene-3,IR:Inhibitory receptors, APCs: antigen presenting cell.

Correlation Between Cervical Cancer and IRs Expression

Correlation Between Cervical Cancer and PD-L1 Expression

As mentioned, papillomaviruses stimulate cell proliferation and variation through oncoproteins, regulate cell survival, and lead to morbidity and cancer progression. PD-1, expressed on the surface of activated T cells, interacts with PD-L1/2, which is located on the surface of tumor cells or immune cells (antigen-presenting cells). The negative regulation of PD-1 pathway leads to T cell failure. The interaction of PD-L1 and CD80 on activated T cells also led to a weakening of cytotoxic T cells. T cell depletion, T cell tolerance, effector T cell response, and T cell activation are all regulated by PD-1 inhibitory receptors.^19,20 In HPV-associated head and neck squamous cell carcinoma (HNSCC), high expression of PD-L1 was observed on tumor cell membranes accompanied by up-regulation of IFN-γ mRNA expression. These results suggest that HPV infection promotes IFN-γ secretion and induces PD-L1 expression. ²¹ Subsequently, several research groups studied whether HPV infection affects the expression of PD-L1 in cervical cancer, and the results showed that HPV-positive status was positively correlated with an increase in PD-L1 expression. The PD-1/PD-L1 pathway leads to cytotoxic CD8 + T-cell dysfunction, a barrier to cancer treatment, and causes tumor cells to escape from the immune system. ²² PD-L1 is expressed on the surface of cervical cancer tumor cells, antigen-presenting cells, and TILs. In cervical cancer stroma, PD-1 positive cells are mostly T cells. The expression of PD-1 in the cervical cancer stroma has been observed in 46.9%∼60.8% of patients. ²³ In addition, PD-L1 + tumor-associated macrophages in patients with cervical adenocarcinoma are associated with relatively low disease-free survival. The expression level of PD-1 on T cells increased with the progression of cervical intraepithelial neoplasia from cervical intraepithelial neoplasia (CIN) I to CIN III, and the expression of PD-L1 on dendritic cells also increased. ²⁴ Moreover, the cytokine profile transforms in cervical cancer from Th1 (containing interferons and IL-12) to Th2 (containing IL-10), which has a more potent immunosuppressive effect. Therefore, the decrease in interferon levels observed during the progression of cervical lesions may be related to the lower expression of PD-L1 in invasive SCC than in CIN I-II. In 2018, Feng et al. used 54 patients with cervical HPV-16 infection and normal cervical cytology as the two experimental groups to determine the correlation between PD-L1 expression and HPV-induced cancer. They found precancerous cervical lesions have higher PD-L1 levels than normal cervical cells. Overexpression of PD-L1 leads to persistent HPV infection in cervical precancerous lesions. ²⁵

Other researchers have studied PD-L1 expression in cervical cancer tissues. Histological analysis showed that PD-L1 was expressed in 80% of the cervical SCC cases. PD-L1 expression is not found in normal cervical tissues but is found in 95% of CINI-II. The expression of PD-L1 in cervical SCC varies widely, ranging from 19% to 22%, and 51% to 88%. PD-L2 was expressed in 29% of cervical cancer cases. These differences may be owing to the use of different critical thresholds and measurement methods. ²⁶ Compared to SCC, the expression of PD-L1 in cervical adenocarcinoma was lower (14% vs 54%). Similar results were observed in lung cancer, where the rate of PD-L1 expression in tumor cells was approximately 52% in SCC and approximately 17% in adenocarcinoma, for reasons that remain unclear. The clinicopathological features appear to be independent of PD-L1 expression in cervical cancer. However, survival analysis showed that patients with cervical SCC with high PD-L1 expression had lower survival rates than those with low PD-L1 expression. ²⁷

In The (TCGA) database, PD-L1 amplification was observed in 22% of patients with cervical SCC. In addition, PD-L1 can be expressed on TIL, indicating that it plays a blocking role in the antitumor response. In a study of cervical SCC samples, the expression rates of PD-L1 in cancer cells and TIL were 59.1% and 47.0%, respectively. ²⁸ These data indicate that PD-L1 and PD-1 are widely expressed in cervical cancer cells and stroma, suggesting that PD-1/PD-L1 inhibitors are potential therapeutic targets. Table 2 summarizes published clinical trials evaluating the efficacy of PD-1/PD-L1 in the treatment of HPV-associated tumors.^21,29–32 In addition, two clinical trials have achieved satisfactory clinical results (Table 3). Pembrolizumab, a monoclonal antibody that blocks the expression of PD-1 receptor on T cells, is expected to be a second-line treatment for patients with PD-1-positive cervical cancer. Open-label phase II, multi-cohort KEYNOTE-158 trial investigated the safety and clinical efficacy of pembrolizumab in the treatment of advanced cervical cancer. The Check Mate 358 study explored nivolumab, another monoclonal antibody that inhibits the PD-1 receptor. This is a Phase I/II study in patients with virus-associated tumors, including patients with recurrent or metastatic cervical, vaginal, or vulvar cancer.^29–31

Table 2.

Clinical Trial Evaluating the Role of ICIs in HPV-Associated Cancers.

Drug name	Drug type	Cancers	Response rates	Reference
Pembrolizumab	Anti-PD-1/PD-L1	R/M cervical cancer	17%	29,30
Nivolumab	Anti-PD-1/PD-L1	GYN cancers	20.8%	31
Nivolumab	Anti-PD-1/PD-L1	SCCA	24%	32
Pembrolizumab	Anti-PD-1/PD-L1	RM HNSCC (HPV-positive, HPV-negative)	18% (25%,14%)	33
Durvalumab	Anti-PD-1/PD-L1	RM HNSCC (HPV-positive, HPV-negative)	16.2% (29.4%, 10.8%)	21

PD-1: Programmed cell death protein-1, PD-L1/2: Programmed death ligand 1/2, R/M: recurrent and/or metastatic, GYN cancers: cervical, vaginal, and vulvar cancers, SCCA: squamous cell carcinoma of the anal canal, HNSCC: head and neck squamous cell carcinoma.

Table 3.

PD-1/PD-L1 Inhibitors are Used for Second-Line Treatment of Recurrent, Persistent, or Metastatic Cervical Cancer.^30,31

	KEYNOTE-158	Check Mate 358
Treatment	Pembrolizumab	nivolumab
Phase	II	I/II
N	98	19
Stage
II	1(1.0%）	1(5.3%）
III	4(4.1%）	2(10.5%）
IV	93(94.9%）	16(84.2%）
PD-L1 expression
Positive	82(83.7%）	10(62.5%）
Negative	15(15.3%）	6(37.5%）
Unknown	1(1%）	3(15.8%）
ORR	12.2%(6.5 to 20.4)	26.3%(9.1 to 87.4)

PD-1: Programmed cell death protein-1, PD-L1/2: Programmed death ligand-1/2, ORR: Overall Response Rate.

PD-1 inhibitor therapy can reactivate T cells highly responsive to neoantigens on tumor cells to enhance the immune response to cancer. Although PD-1-blocking antibodies have greatly improved the response rate to cancer treatment, three major problems remain. First, some patients may develop autoimmune reactions. A meta-analysis reported that the most common immune-related adverse events associated with pembrolizumab were arthralgia, pneumonia, and liver toxicity; nivolumab was associated with endocrine toxicity; atezolizumab with hypothyroidism; and ipilimumab with skin, gastrointestinal, and kidney toxicity. ³³ Treatment plan was suspended in 10% of pembrolizumab patients and 8.7% of patients with cemipilimab due to adverse reactions.^34–36 Although many attempts have been made to enhance anti-tumor efficacy without increasing side effects, only some have been successful. Second, more than half of the patients did not respond to the treatment. Currently, there are no predictive biomarkers for the response to PD-1 blocking therapy. Little is known about the specific mechanism by which PD-1 blockade inhibits tumor growth. Third, the best combination to maximize the therapeutic effect is yet to be determined.

Correlation Between Cervical Cancer and CTLA-4 Expression

In addition to the molecular expression of PD-1-related pathways, other immunomodulatory molecules have also been found to be expressed in patients with cervical cancer. CTLA-4 was the first therapeutically targeted immune checkpoint receptor. It is only expressed on T cells and can downregulate the function of T cells to prevent the overactivation of the immune system.^37,38 CTLA-4 regulates the establishment of neonatal immune responses, whereas the PD-1 pathway primarily influences sustained immune responses. The biological effects of CTLA-4 and PD-1 are utilized in different parts of the body and during specific periods of the T-cell life cycle, where these receptors functionally complement each other and regulate the balance between T-cell immune tolerance to self-antigens and proliferation in response to foreign antigens. The function of PD-1 is different from that of CTLA-4, which mainly plays a regulatory role in lymphatic organs. PD-1 controls T cell activation in local peripheral tissues. ³⁹ CTLA-4 inhibits the costimulatory signals required for full T-cell activation after interaction between the T cell receptor and the antigen/MHC on the antigen-presenting cell. Studies have found that compared to the control group, CTLA-4 expression is increased and CD28 expression is decreased in the peripheral blood mononuclear cells of patients with advanced cervical cancer, ⁴⁰ thus transforming the immune response into stronger immune tolerance. Blocking CTLA-4 can activate CD4 + and CD8 + effector cells in tumors, selectively depleting regulatory T cells in tumors, thereby strengthening anti-tumor immunity and tumor rejection. Some genetic polymorphisms of CTLA-4 have been shown to be associated with an increased risk of cervical cancer. CTLA-4 expression is found in more than 50% of invasive cervical cancer cells, and is related to the clinical phase of the tumor and lymph node metastasis. ⁴¹ A clinical trial found that the use of ipilimumab (anti-CTLA-4) after radiotherapy and chemotherapy in patients with locally advanced cervical cancer induced the proliferation of central memory T cells and effector memory T cells. ⁴² Therefore, inhibiting CTLA-4 can potentially help patients fight cervical cancer. The rationale for using anti-CTLA4 antibodies in cancer immunotherapy is to release a pre-existing anti-cancer T cell response and potentially trigger a new T cell response by inhibiting CTLA-4's restriction on the formation of nascent immune responses. However, most adverse events are mild. According to the report, the incidence of serious adverse events caused by the combination of anti-CTLA-4 and anti-PD-1 drugs was 55%, ⁴³ which was significantly higher than that of drug alone. A third of these patients were forced to stop treatment. More clinical trials are needed to explore the use of CTLA in cervical cancer to minimize side effects and identify biomarkers that can predict treatment effectiveness.

Correlation Between Cervical Cancer and TIM-3 Expression

Another immunosuppressive molecule is TIM-3. TIM-3 is also known as Hepatitis A virus cell receptor 2 (HAVCR2) or CD366. This protein is composed of a cytoplasmic C-terminal tail and a single transmembrane domain signal TIM-3, which plays an important role in immune detection.^44,45 It is expressed on the surface of T cells and innate immune cells. It has four ligands, the main one of which is galectin-9. TIM-3 is mainly expressed in CD8 + T cells, indicating dysfunction and immunosuppression, and is often co-expressed with PD-1 to reduce the secretion of IFN-γ, TNF-α, IL-2, and other factors, thus promoting the failure of effector T cells. ⁴⁶ As shown in preclinical models, dual blocking of PD-1 and TIM-3 resulted in a significantly enhanced T cell response compared to the single blocking of PD-1.^47–49 TIM-3 has had a limited effect as a single immunotherapy in clinical trials, but the combination of TIM-3 monoclonal antibodies with other ICIs, such as CTLA-4 and PD-1, enhances the synergistic effect. In addition, unlike previously identified immune checkpoints, TIM-3 expression is limited to differentiated T cells and intra-tumoral Tregs, thus avoiding the side effects caused by CTLA-4 ⁵⁰ and PD-1 blockade.^51,52 It has also been reported that TIM-3 + Tregs cells have stronger immunosuppressive effects than TIM-3-Tregs cells.TIM-3 blockers thus restore anti-tumor CD8 + T cell response and mediate Treg depletion. TIM-3 is expressed on Treg cells, and in a few patients with cervical cancer (n = 3), up to 60% of TIL are CD4 + TIM3 + cells, which is an apparently higher proportion than non-invasive T cells. ⁴⁴ A large number of scientific studies have shown that the overexpression of TIM-3 may be associated with poor survival in solid tumors and may be associated with more severe or advanced disease.^53–55 Some in vitro studies have shown that TIM-3 expression is associated with cancer cell metastasis and invasion.

In addition to TIM-3 expression in T cells, TIM-3 is also shown to be expressed in tumor cells. Increased TIM-3 expression has been detected in various cancers, including cervical cancer, prostate cancer, osteosarcoma, and esophageal adenocarcinoma. High TIM-3 expression is associated with poor clinical outcomes. ⁵⁶ Overexpression of TIM-3 is related to HPV positivity, so it is also associated with low differentiation of cervical cancer tumors and short survival time.^57,58

Co-expression of TIM-3/Gal-9 and PD-1/PD-L1 has been found in cervical cancer. ⁵⁷ TIM-3 is expressed positively in 15% of chronic cervicitis, 50% of cervical CIN, and 65% of cervical cancer. ⁵⁴ It is also associated with cervical cancer stage, lymphatic metastasis and disease progression. The 5-year survival rate was 46.4% in TIM-3 positive group and 80% in TIM-3 negative group (P = 0.006). TIM-3 expression was found in cytoplasm of Siha and Hela cell lines in vitro. TIM-3 knockdown can reduce the migration and invasion of Hela cells. However, only a few studies have been conducted on cervical cancer. Therefore, the efficacy of TIM-3 blocking in the treatment of cervical cancer requires further investigation.

Correlation Between Cervical Cancer and TIGIT Expression

TIGIT is a member of the poliovirus receptor (PVR, CD155) connexin family. It is expressed in CD4 + and CD8 + T cells, Tregs, and NK cells. Its primary ligand is PVR, which is overexpressed in tumor cells and tumor-associated myeloid cells.^59,60 Other proteins such as CD112 and CD113 also interact with this receptor. The binding reaction can inhibit T-cell activation. ⁶⁰ The TIGIT-PVR axis has a different mechanism of action from classical immune checkpoints, making it an attractive target for immunotherapy. Unlike PD-1 and CTLA-4, NKs constitutively express TIGIT, and its expression in tumors is up-regulated. ⁶¹ Thus, anti-TIGIT therapy has shown a unique ability to simultaneously target two major types of anti-tumor effector cells. Dual blockade of TIGIT and PD-1 may restore and improve immune cell activity in cervical cancer. For example, it has been reported that different subsets of T cells expressing both PD-1 and TIGIT are significantly increased in the peripheral blood of patients with cervical cancer. ⁶² Similar findings have been found in cervical cancer. CD96 was recently found to be expressed on T and NK cells of cervical cancer, and it is another member of the TIGIT axis. Together with PD-1, it attenuates the function of CD8 + tumor-infiltrating lymphocytes. Therefore, the dual blocking of CD96 and PD-1 significantly enhanced the function of CD8 + tumor-infiltrating lymphocytes and inhibited tumor growth in mouse cervical cancer models. ⁶³ Tiragolumab is a humanized antibody that blocks the binding of TIGIT to its ligand (CD155) to enhance the activity of T and NK cells. Tiragolumab is being investigated in the Phase II SKYSCRAPER 04 (NCT04300647) trial. The trial compared atezolizumab with tiragolumab in patients with metastatic and/or recurrent PD-L1-positive cervical cancer. ⁶⁴ Although there are currently only a few preclinical and clinical studies involving immunotherapy for TIGIT and cervical cancer, we expect encouraging results.

Correlation Between Cervical Cancer and LAG-3 Expression

In 1990, Triebel and colleagues identified a type I transmembrane protein, lymphocyte Activation Gene-3 (CD223), consisting of 498 amino acids, on activated human NK and T cell lines. ⁶⁵ The LAG-3 gene is located on human chromosome 12 (mouse chromosome 6), adjacent to CD4. LAG-3 is highly homologous and closely related to CD4 in structure, with four extracellular immunoglobulin superfamily (IGSF)-like domains (D1-D4). Owing to its highly conserved structural elements, LAG-3 can bind to MHC-II molecules with a stronger affinity than CD4. ⁶⁶

The main ligand of LAG-3 is a molecule of the major histocompatibility complex (MHC) class II, which binds to the conserved extension ring of the LAG-3 D1 domain. Once bound to the MHC II class, LAG-3 transmits inhibitory signals through its cytoplasmic domain to inhibit CD4 + T cell activation. LAG-3 also contributes to the escape of tumors from apoptosis; however, its signal transduction mechanism remains unclear. For example, in human melanoma cells, increased expression of MHC class II molecules is associated with poor prognosis in patients. LAG-3 binding to MHC class II contributes to tumor escape from apoptosis and tumor-specific CD4 + T cell recruitment and subsequently reduces CD8 + T cell response. There are other ligands for LAG3. Galectin-3 is expressed on cells other than tumor cells in the tumor microenvironment; therefore, its interaction with LAG-3 on tumor-specific CD8 + T cells may mediate antitumor immune responses. LSECtin, expressed in the liver and found in human melanoma tissues, promotes growth by inhibiting anti-tumor T-cell-dependent responses. ⁶⁷ Interactions between these two potential replacement ligands may amplify the effect of LAG-3 on T cell function, especially the significant role that LAG-3 plays in CD8 + T cells in the tumor microenvironment.

Wang et al ⁶⁸ found that FGL-1 could be a potential ligand for LAG-3. FGL-1 is secreted by liver cells under normal physiological conditions. It is a member of the fibrinogen family and has a similar structure to fibrinogen but has no definite role in platelets or thrombosis. However, some tumor cells secrete FGL-1 in large quantities and are associated with poor prognosis and immunotherapy resistance. Current anti-LAG-3 antibodies may also be required to target the LAG-3/FGL-1 interactions to reactivate depleted T cells fully. Just like PD-1 and CTLA-4, LAG-3 is not expressed on naïve T cells but mainly on activated T cells, NK cells ⁶⁹ and plasma dendritic cells. Antigen stimulation induces the expression of CD4 + and CD8 + T cells. Under physiological conditions, LAG-3 is the activation marker of CD4 + and CD8 + T cells, which can be detected 24 h after stimulation in vitro; its expression peaks 48 h later and decreases on the eighth day in mice. The secretion of IL-2, IL-7, IL-12, TNF, and IFN-γ increases LAG-3 expression in activated T cells. Zhang et al ⁷⁰ demonstrated that LAG-3 is present in Tregs and limits their proliferation. However, the role of LAG-3 in the effector functions of Tregs remains controversial. Natural regulatory plasma cells that produce IL-10 express LAG-3. The study results of Fucikova et al showed a strong correlation between the density of CD8 + T cells infiltrated by tumors, the number of tumor cells, and the existence of a large number of LAG-3 + cells in tumors, suggesting that LAG-3 acts as a marker of T cells in the tumor environment. ⁷¹ In vitro proliferation tests have also demonstrated that LAG-3 expression on Tregs is required for maximum inhibitory activity, as blocking LAG-3 results in the loss of Treg function. Furthermore, anti-LAG-3 blocked antigen-specific CD4+ Treg-mediated protection in an in vivo model of pulmonary vasculitis. Therefore, LAG-3 is crucial for Treg cell function. In addition, the transfection of LAG-3 into non-Treg CD4 +T cells resulted in the acquisition of a regulatory phenotype and decreased the proliferation of co-cultured reactive T cells. Recent studies have shown that LAG-3 promotes Treg differentiation, while LAG-3 blocking inhibits Treg induction. The effect of LAG-3 on NK cells is unclear; however, the proliferation of activated NK cells is reduced by LAG-3 signal transduction, leading to cell cycle arrest in the S phase (Figure 1).^72–74 LAG-3 is expressed in various HPV-associated malignancies, especially in cervical cancer with an expression rate of up to 75%. ⁷³ WT1 plays a tumorigenic role in the process of carcinogenesis and has been found to be expressed in various solid tumor cells such as cervical cancer cells. LAG-3 blocking significantly increased the expression of tumor antigen-specific T cells in WT1, while PD-1 blocking was not significant. ⁷⁵

Figure 1.

Roles of LAG-3 in CD + 4 cells, CD + 8 cells, Treg cells and DC cells in tumor microenvironment.

As a consequence, adding LAG-3 blockers to the treatment of blocking PD-1 can better activate anti-tumor immunity, and further improve the combined treatment effect of LAG-3 and PD-1 blocking. Furthermore, LAG-3 blocking can inhibit Treg cells with suppressive function, and its effect on Treg cells has been observed in mouse models of both LAG-3 and PD-1 deletions. Multiple studies have shown that double-blocking LAG-3 and PD-1 can enhance the proliferation of T cells and increase the number of effector T cells compared with blocking PD-1 alone. Improve the ability of T cells to kill, thereby inhibiting tumor growth.LAG-3 is also expressed on Tregs in the peripheral blood and tumor tissues of patients with melanoma, colorectal cancer, and non-small cell lung cancer. These LAG-3 expressing Treg cells produce high levels of the IL-10 and transforming growth factor-β (TGF-β), thereby inhibiting tumor-specific T cells. ⁷⁶

In addition, in various types of human tumors, the expression level of LAG-3 and the infiltration level of LAG-3 + cells are correlated with tumor progression, prognosis, and outcome. Cervical cancer tissue samples, particularly those associated with HPV, showed high expression of LAG-3. Matsuzaki et al observed that about 80% of LAG-3 + and 50% of LAG-3− tumor infiltrating CD8 + T cells expressed PD-1, respectively. The expression of LAG-3 was detected in cervical cancer TIL with an intensity ranging from 100 to 57.5%, and an association between LAG-3 and PD-1 expression was also found. In squamous cell carcinoma and adenocarcinoma of the cervix, elevated LAG-3 levels are correlated with the co-expression of PD-1. ⁵⁴ LAG-3 + tumor-infiltrating lymphocytes were observed in 91% of the vulvar squamous tumors. ⁷⁷ Many monoclonal antibodies targeting LAG-3, most of which are in clinical trials, such as IMP321(the first LAG3 fusion protein) and relatlimab(the first LAG3 monoclonal antibody), show great potential in tumor immunotherapy. ⁷⁸ However, the therapeutic effect of cervical cancer remains to be studied.

Recently, research elucidated that the FGL-1/LAG-3 pathway is another immune checkpoint pathway that plays a vital role in the immune escape mechanism, which is similar to the PD-1/PD-L1 pathway and may mediate resistance to PD-1/PD-L1 therapy. As a ligand of LAG-3, FGL-1 is a valuable biomarker for predicting PD-1/PD-L1 resistance. FGL-1 is the main binding protein of the LAG-3 antibody, and their interaction is stable, specific, and conserved across species. Further domain deletion studies showed that the F protein domain of FGL-1 and the D1-D2 domain of LAG-3 were involved in independent interactions between FGL-1 and LAG-3. ⁶⁸ In vivo, experiments showed that the FGL-1/LAG-3 interaction was inhibited by gene knockout or antibody blocking, whereas anti-tumor immunity was promoted by stimulating the activation and expansion of TIL. Although the specific mechanism by which FGL-1/LAG-3 regulates T-cell function remains unclear, studies have shown that blocking FGL-1 can synergistically inhibit PD-1/PD-L1 signal transduction with anti-PD-1/PD-L1. This conclusion was on the basis of previous research on the synergistic inhibitory activity, which has been confirmed in animal models. ⁷⁹ Because of the high affinity between FGL-1 and LAG-3, FGL-1/LAG-3 and PD-1/PD-L1 can independently regulate T cells, and blocking these two checkpoints can produce synergistic anti-tumor effects. As mentioned previously, LAG-3 is an effective therapeutic target for cancer immunotherapy.

Conclusion

Immune checkpoints are important anti-tumor response targets for advanced metastatic cervical cancer patients. Despite the potential advantages of immunization strategies, challenges remain. We found the following limitations. First, many patients have yet to respond to these treatments, even in sensitive tumor types. Second, because many patients experience side effects, it remains challenging to determine which patients are suitable for this therapy and have promising results. Third, the best opportunity to apply immunotherapies and how to add them to existing treatment algorithms needs to be studied. LAG-3 is considered a promising target that exerts immunomodulatory effects on several processes involved in carcinogenesis. A growing number of preclinical studies have shown that combining targeted-blocking antibodies with other immune drugs increases patients’ antitumor response. Therefore, inhibitory receptors are expected to be a new target for cancer treatment.