Progress of immune checkpoint inhibitors in gastric cancer

Abstract

Gastric cancer (GC) is one of the most common malignant tumors globally. The screening rate of GC is low, and the early symptoms are not obvious, which affects early diagnosis, and most patients have a poor prognosis. Various treatments are available for GC, mainly surgery, chemotherapy, radiotherapy, targeted therapy and immunotherapy. Immunotherapy has made significant progress in recent years, especially for unresectable and metastatic GC. Immune checkpoints are proteins which can regulate the congenital and adaptive immunity, and are mainly expressed on immune cells. Immune checkpoints are crucial molecules that regulate the immune system, maintaining immune balance through positive or negative modulation. Tumors exploit negative immune checkpoint molecules to suppress immune responses, thereby evading immune surveillance. Immune checkpoint inhibitors (ICIs) can undo this inhibition, reactivating immune cells to destroy tumor cells. The prospects for the treatment of GC with ICIs are promising, but it also faces many difficulties and challenges. This minireview summarizes the progress of immune ICIs in GC, discusses current individualized strategies, and explores future development directions.

Key Words: Gastric cancer; Immune checkpoint inhibitors; Combination therapy; Chemotherapy; Targeted therapy

Core Tip: Gastric cancer (GC) is a serious threat to human health. Immunotherapy is one of the important ways of clinical treatment of GC. Among them, immune checkpoint inhibitors (ICIs) have been applied in clinical practice, and the research on the combination therapy of ICIs has shown good therapeutic effect and lower treatment toxicity. This minireview summarizes the progress of immune ICIs in GC, discusses current individualized strategies, and explores future development directions.

INTRODUCTION

Gastric cancer (GC) is one of the most frequent malignant tumor of the digestive tract. It is also the fifth most common cause of cancer-related death worldwide[1]. Causes of GC include Helicobacter pylori infection, unhealthy dietary and lifestyle habits, Epstein-Barr virus (EBV) infection, and genetic risk factors[2,3]. The early symptoms of GC are non-specific and do not draw patients' attention. With the popularization of gastroscopy, the incidence of GC has decreased, but most patients are diagnosed too late and lost the opportunity for resection. Due to the ineffectiveness and the emergence of multidrug resistance, the prognosis for patients remains poor, with a 5-year survival rate of less than 5%[4]. Systemic treatment is an effective method for prolonging survival in patients with metastatic or unresectable GC. Systemic treatment includes immunotherapy, targeted therapy, and chemotherapy. Immunotherapy has demonstrated favorable therapeutic effects for advanced metastatic GC. Immunotherapy complements surgery, chemotherapy or radiotherapy, bringing new hope to patients with advanced and metastatic GC.

IMMUNE CHECKPOINT INHIBITORS

The main cause of tumor is the cells evade the body's immunologic system, enabling the growth and invasion of the tumor. Therefore, blocking tumor escape from immune examination is an important immunological strategy. Immune checkpoints are important molecules and key nodes in the immune regulatory pathway, including inhibitory receptors and ligands[5]. Immune checkpoints can regulate the immune responses to disrupt cells. Immune checkpoint proteins are mainly expressed on antigen-presenting cells (APCs), the ligand proteins expressed on cancer cells. When the immune checkpoint binds to the ligand, it will send an inhibitory signal to control T cell activation[6]. Immune checkpoint inhibitors (ICIs) can prevent checkpoint proteins from binding to their ligand, and the inhibitory signals will be blocked. ICIs can lift this inhibitory effect, reactivating immune cells to resume their function in destroying tumor cells[7].

Activated T cells can express surface inhibitory receptors such as cytotoxic T lymphocyte-associated protein 4 (CTLA-4) (CD152) and programmed death 1 (PD-1) (CD279). Many tumor cells evade T cell-mediated destruction by expressing ligands such as programmed death ligand 1 (PD-L1) (CD274, B7 homolog-1), which are recognized by the PD-1 T cell receptor[8]. The Food and Drug Administration (FDA) has approved multiple ICIs for the clinical treatment of various cancers. The ICI drugs that have been marketed include PD-1 inhibitors (Cemiplimab, Nivolumab, Pembrolizumab, Dostarlimab, Retifanlimab, Toripalimab), PD-L1 inhibitors (Durvalumab, Avelumab, Atezolizumab), CTLA-4 inhibitors (Tremelimumab, Ipilimumab), and lymphocyte activation gene-3 (LAG-3) inhibitor Relatlimab[9]. Many clinical trials have been registered on clinicaltrials.gov for ICI treatment in GC to evaluate the efficacy of single-agent, combination regimens of multiple ICIs, and combined with other drugs (Table 1)[10-14]. Extensive clinical trials have revealed the promising therapeutic effects of ICIs in GC. The FDA has approved several PD-1 inhibitors as monotherapy or combinations for GC, which have been recognized by clinicians and patients.

Table 1 Immune checkpoint inhibitors approved by the Food and Drug Administration for gastric cancer.

Drug	Brand name	Target	Medication regimen	Indication	Ref.
Nivolumab	Opdivo	PD-1	Alone	Stage II/III GC/GEJ with residual pathological tissue after neoadjuvant chemoradiotherapy	Kelly et al[10]
Nivolumab	Opdivo	PD-1	Nivolumab combined with chemotherapy (leucovorin, fluorouracil, and oxaliplatin or capecitabine and oxaliplatin)	Advanced metastatic HER-2-negative GC/GEJ	Janjigian et al[11]
Pembrolizumab	Keytruda	PD-1	Pembrolizumab combined with chemotherapy (cisplatin and 5-FU)	Locally advanced metastatic GC/GEJ	Sun et al[12]
Pembrolizumab	Keytruda	PD-1	Pembrolizumab combined with chemotherapy (platinum and fluoropyrimidine) and Trastuzumab	Locally advanced metastatic unresectable HER-2-positive GC/GEJ	Janjigian et al[13]
Pembrolizumab	Keytruda	PD-1	Pembrolizumab with chemotherapy (oxaliplatin with capecitabine or cisplatin and 5-FU)	Locally advanced metastatic HER-2-negative GC/GEJ	Rha et al[14]

GC/GEJ: Gastric cancer/gastroesophageal junction; HER-2: Human epidermal growth factor receptor 2; PD-1: Programmed death 1; 5-FU: 5-fluorouracil.

CTLA-4 inhibitors

CTLA-4 is a transmembrane receptor on T cells and is highly homologous to CD28 on T cells[15]. CTLA-4 competitively inhibits the interaction between B7 and CD28, and suppresses the T-cell activation pathway mediated by CD28. It then transmits inhibitory signals to hinder T cell activation, and tumor cell immune escape (Figure 1)[16]. CTLA-4 can reduce the functional defects of normal tissues and weaken the autoimmune response by a negative feedback role[17]. CTLA-4 inhibitors can reduce immunosuppressive regulatory T cells (Tregs) in the tumor microenvironment (TME), and the anti-tumor immune response will be enhanced[18]. At present, a number of CTLA-4 inhibitors have been used in clinical cancer treatment and showed good therapeutic effect, but the effect was not ideal in clinical studies of GC.

Figure 1 Mechanism of immune checkpoint inhibitors. Blocking programmed death 1, programmed death ligand 1, and cytotoxic T lymphocyte-associated protein 4 can activate T cells, leading to the death of tumor cells. APC: Antigen-presenting cell; CTLA-4: Cytotoxic T lymphocyte-associated protein 4; GC: Gastric cancer; MHC: Major histocompatibility complex; PD-1: Programmed death 1; PD-L1: Programmed death ligand 1; TCR: T cell receptor.

Ipilimumab is a CTLA-4 inhibitor, which is a human monoclonal antibody (mAb) [immunoglobulin G (IgG) 1]. It promotes anti-tumor responses by activating T cells and increasing tumor infiltration. In 2011, the FDA approved Ipilimumab to treat unresectable or metastatic melanoma in the clinic[19]. Ipilimumab showed good therapeutic effect on melanoma and attracted the attention of clinicians to research its curative effect on other solid tumors. The safety and efficacy of Ipilimumab monotherapy in advanced metastatic GC was evaluated in a phase II clinical study[20]. The study showed that Ipilimumab monotherapy did not improve progression-free survival (PFS) in GC, but CD4+ T cells in the circulating blood were increased. In addition, treatment-related adverse events (TRAEs) mainly manifested as pruritus (31.6%) and no deaths related to study treatment were observed. The clinical effect of sequential or maintenance monotherapy with Ipilimumab for GC was not ideal, but it showed good safety. To improve the treatment effect of Ipilimumab against GC, further research is required.

Tremelimumab (CP-675, 206) is a fully humanized CTLA-4 mAb of the IgG2 isotype, which was designed to reduce complement activation and lower the risk of cytokine storm[21]. Compared with Ipilimumab, it has a longer half-life and the administration time can be extended to once every three months. A phase II clinical trial of Tremelimumab for the treatment of metastatic GC showed that the median overall survival (OS) time was 4.83 months. However, evidence of disease control was observed in a small group of patients, as assessed by regular computed tomography scans (4 out of 18 cases) or the decline or stabilization of serum tumor-associated antigen (5 out of 18 cases), suggesting clinical benefit[22]. The results of this experiment were not satisfactory, with only one patient achieving significant and lasting benefits. The most common side effects were immune-mediated, with relatively mild itching (in 50% of patients), diarrhea and fatigue (both 28%). Despite the unsatisfactory results of this study, most patients tolerated Tremelimumab well during the treatment period, even though their symptoms worsened. This study had some limitations, including the lack of long-term outcome analysis, potential bias and a moderate level of evidence.

CTLA-4 inhibitors were the first ICIs approved for clinical cancer treatment, and have revolutionized cancer therapy. However, therapeutic efficacy in GC is not satisfactory. Researchers have explored the mechanisms that limit the effectiveness of CTLA-4 inhibitors. The reasons for the anti-tumor limitations of CTLA-4 inhibitors may be due to T cells in the TME. Effector T (Teff) cells and Tregs have been proved to mediate the different therapeutic activity of CTLA-4 inhibitors in the TME. Tregs regulate cessation of the immunologic system to maintain stability, and inhibit anti-tumor immune responses to prevent excessive immune response. Teffs are the main effector cells in the body, which affect anti-tumor immunity and anti-infection immunity[23,24]. The surface of Tregs can express high levels of CTLA-4; therefore, CTLA-4 inhibitors will impair the suppressive activity of Tregs[25].

This limitation might be related to the Fc fragment. CTLA-4 inhibitors reduce Tregs in the TME. This effect may be related to the Fc fragment of the CTLA-4 inhibitor. The Fc fragment of CTLA-4 inhibitors binds to FcγR which is on the surface of tumor-infiltrating APCs/macrophages. The antibody-dependent cell-mediated cytotoxicity (ADCC) will then be activated to eliminate Tregs, and this is the key to exerting anti-tumor activity[26]. The Fc receptor (FcR) is a type of protein on the surface of the cells that can specifically bind to the Fc fragment of antibodies, and its surface contains a chain (FcγR) that transmits FcR signals. FcγR IIb is one of the subgroups of FcγR[27]. FcγR IIb has an immunoreceptor tyrosine-based inhibitory motif structure in its intracellular domain, so only FcγR IIb mediates immunosuppressive signals and downregulates the corresponding functions of cells after activation[28]. FcγR IIb is widely present in various immune cells, and is especially highly expressed in the TME. Therefore, Ipilimumab or Tremelimumab cannot effectively eliminate Tregs, and their anti-tumor effects are mainly by CD8+ T cells. This may explain why CTLA-4 inhibitors are ineffective in GC treatment. Based on the therapeutic effect of CTLA-4 inhibitors in melanoma, enhancing the anti-tumor effect in GC is a future research direction. Firstly, CTLA-4 inhibitors can be combined with other drugs to enhance the therapeutic effect through the interaction between drugs and complementary targets. On the other hand, FcγR IIb can serve as an important immune checkpoint in the TME. By adding an antibody targeting FcγR IIb to the Fc fragment, the Fc-mediated depletion of Tregs within the tumor can be enhanced[29]. CTLA-4 inhibitors bring hope to GC patients, and clinical trials of multiple combination strategies for GC are ongoing.

PD-1 inhibitors and PD-L1 inhibitors

PD-1 is another representative immunosuppressive checkpoint following the discovery of CTLA-4[30]. PD-1 is mainly expressed on the surface of immune cells, include activated CD4+ and CD8+ T lymphocytes, dendritic cells (DCs), B lymphocytes, and so on[31,32]. PD-L1 and PD-L2 are both ligands of PD-1. PD-L1 is the primary ligand expressed on immune and tumor cells, playing a role in immune escape. PD-L2 is associated with immune tolerance which can be expressed in immune cells, stromal cells, and tumor cells. While PD-1 binds to PD-L1 or PD-L2, T cell receptors and CD28 signal transduction will be inhibited by activating SHP-2 phosphatase. The PD-1/PD-L1 pathway inhibits the Ras-Raf-MEK-ERK and phosphatidylinositol 3-kinase (PI3K)-protein kinase B (AKT) signaling pathways to impair T cell activation and cytokine production[33,34]. These cytokines mainly include interferon-γ (IFN-γ), tumor necrosis factor, interleukin-2 (IL-2), and other cytokines to inhibit cellular and humoral immunity[35]. The malignancy grade of GC can be elevated by the level of PD-L1 expression. The inhibitors of PD-1/PD-L1 bind to PD-1 protein of immune cells or PD-L1 molecules produced by tumor cells. Thus, PD-1 cannot bind to PD-L1, thus enhancing the immune system and killing tumor cells (Figure 1).

PD-1 inhibitors have shown good therapeutic effects in the treatment of GC, and a variety of combination strategies have been tried to further improve the therapeutic effect in GC. The major challenges of PD-1/PD-L1 inhibitors include immune-related adverse events of varying degrees, including cytokine release syndrome, cardiotoxicity, pneumonia, hepatitis, endocrine dysfunction, fatigue, rash and diarrhea. However, their toxic reactions are relatively controllable, and significant improvement can be achieved by discontinuing the drug or symptomatic treatment[36]. Although immunotherapy is a long-term therapy, drug resistance can also develop. Researchers have conducted many clinical studies on advanced metastatic GC, and single-agent PD-1 inhibitors have shown controllable safety and good anti-GC activity (Table 2)[37-45]. However, their therapeutic effect does not show superiority compared with first-line chemotherapy. Future research needs to explore which GC patients can achieve the greatest potential benefits from ICIs. Combination therapies with other immunotherapies, radiotherapy and chemotherapy should be explored to achieve the optimal therapeutic effect, the lowest toxic reaction and the lowest drug resistance in GC treatment.

Table 2 Clinical trials of programmed death 1 for gastric cancer.

Immune checkpoint inhibitor	Patient	Method	Phase	Ref.
Pembrolizumab	Recurrent or metastatic PD-L1-positive GC/GEJ	Intravenously once/2 weeks in 2 years or until progression or unbearable toxic reactions	Ib	Muro et al[37]
Pembrolizumab	Previously treated GC/GEJ	Intravenously once/3 weeks until progression, or unbearable toxic reactions	Ⅱ	Fuchs et al[38]
Pembrolizumab	Advanced GC/GEJ with poor outcomes after chemotherapy	Once/3 weeks for 24 months	III	Shitara et al[39]
Pembrolizumab	Untreated, advanced GC/GEJ with positive score (CPS) ≥ 1	Once/3 weeks	Ⅲ	Shitara et al[40]
Camrelizumab (SHR1210)	Recurrent, metastatic refractory or intolerant GC/GEJ after previous chemotherapy	Intravenously once and 4 weeks later once/2 weeks	I	Huang et al[41]
Pembrolizumab	Advanced PD-L1-positive GC/GEJ (CPS ≥ 1)	Intravenously once/3 weeks for 2 years	I	Chung et al[42]
Toripalimab	Advanced chemo-refractory GC patients	Monotherapy with Toripalimab (3 mg/kg, every 2 weeks)	Ib/II	Wang et al[43]
Nivolumab	Locally advanced or metastatic chemotherapy-refractory GC	Intravenously once/2 weeks	I/II	Janjigian et al[44]
Nivolumab	Advanced GC/GEJ, previously treated with chemotherapy	Intravenously once/2 weeks until progression or unbearable toxic reactions	Ⅲ	Kang et al[45]

CPS: Combined positive score; GC: Gastric cancer; GC/GEJ: Gastric cancer/gastroesophageal junction; PD-L1: Programmed death ligand 1.

LAG-3 inhibitors

During the current application of ICIs, it has been found that some patients have developed drug resistance. To overcome this resistance, alternative immune checkpoints are currently being studied, such as LAG-3 (CD223). LAG-3 can negatively regulate T cells and limit the activation of T cells, thereby preventing autoimmunity. LAG-3 is homologous to CD4 and is predominantly expressed in immune cells, such as Tregs, Teffs, activated B cells, natural killer cells, and plasmacytoid DCs[46]. LAG-3 has four ligands, including major histocompatibility complex class II (MHC-II), liver sinusoidal endothelial cell lectin, fibrinogen-like protein 1 and galectin-3[47,48]. LAG-3 and CD4 share the common ligand MHC-II, and the binding affinity of LAG-3 to MHC-II is higher than that of CD4[49]. When LAG-3 competitively binds to MHC-II, the cytokine secretion levels and proliferative capacity of CD4+ T cells will be reduced. The activation, proliferation, and cytotoxicity of CD4 and CD8 T lymphocytes will subsequently be suppressed[50,51]. LAG-3 inhibitors can alleviate this interaction-mediated inhibitory immune response by restoring T cell function and suppressing the activity of Tregs[52]. As one of the new inhibitory checkpoints, LAG-3 is anticipated to emerge as a highly promising target for tumor therapy.

The combination of LAG-3 inhibitors has shown good therapeutic effects. The current study focus is mainly on LAG-3 inhibitors combined with chemotherapy or other ICIs. Blocking LAG-3 and PD-1 Leads to the co-expression of cytotoxic CD8+ T cells to promote anti-tumor immunity in a synergistic manner[53]. The FDA has approved Relatlimab (OPDUALAG) combined with Nivolumab as a novel combination immunotherapy for metastatic melanoma[54]. This combination therapy is a promising approach to modulate immune checkpoint pathways in treating GC[55].

The research of RELATIVITY-060 evaluated treatment effect of Nivolumab combined with Relatlimab and chemotherapy in advanced GC/gastroesophageal junction (GEJ) which was previously untreated. The results indicated that the objective response rate (ORR) was 48% in the combination group (consisting of Nivolumab, Relatlimab, and chemotherapy), and 61% in the group receiving Nivolumab in combination with chemotherapy. However, the median OS and median PFS between the two groups were not significantly different. The rate of TRAEs in the combination group (Nivolumab, Relatlimab and chemotherapy) compared with the Nivolumab plus chemotherapy group was 69% vs 61%[56]. Adding a LAG-3 inhibitor to the current standard treatment (chemotherapy/anti-PD-1 antibody) did not significantly improve the efficacy of advanced GC/GEJ, but safety was acceptable.

Although the addition of LAG-3 inhibitors to GC treatment has not shown satisfactory results, it provides a new combination strategy. How to increase the therapeutic effect of combination therapy in GC is a future research direction. The study of LAG-3 is not yet thorough, and many questions remain to be answered in further research including the following: (1) The signal transduction mechanism of LAG-3 with ligand; (2) Synergistic effect between LAG-3 and other immune checkpoints; and (3) The mechanism with other immune checkpoints still needs further research. The long-term safety and efficacy of LAG-3 inhibitors in GC requires further evaluation.

COMBINATION THERAPY

ICIs combined with chemotherapy

Chemotherapy is an essential treatment strategy in GC. Most chemotherapy drugs are cytotoxic drugs, which cannot distinguish normal cells from cancer cells; thus, they will have some toxicity to normal cells in the body. Therefore, chemotherapy can directly kill tumor cells. In addition, chemotherapy can have an impact on the immune system by inducing immunogenic cell death (ICD), releasing antigens, and disrupting immunosuppressive pathways[57]. The first-line chemotherapy regimen of cisplatin plus fluorouracil has the most ideal effect and the least side effects, which is best for most GC patients[58,59]. Although the current chemotherapy regimens has significantly improved the survival rate of GC, but the OS is less than one year, and the clinical outcomes of GC chemotherapy have yet to reach satisfactory levels[60]. The toxicity and adverse reactions of chemotherapy should not be overlooked. Gastrointestinal and hematological system toxicity, and other side effects are intolerable in many patients.

However, recent studies suggest that chemotherapy has a bidirectional regulatory effect on the TME[61]. Chemotherapy can promote tumor cells to release immunogenic substances and antigen cross-presentation, and enhance the sensitivity of immune cells. This provides a basis for clinical combination therapy and suggests that chemotherapy may enhance the reactivity of ICIs. ICIs can enhance recognition of the immune system to kill tumor cells and improve the anti-tumor response[62]. Theoretically, ICIs combined with chemotherapy can synergistically improve the outcomes of various single-drug treatments[63,64]. Therefore, trials exploring ICIs combined with chemotherapy have shown promising efficacy in clinical treatment (Table 3).

Table 3 Clinical trials of immune checkpoint inhibitors combined with chemotherapy for gastric cancer.

Immune checkpoint inhibitor	Medication regimen	Type of GC	Phase	Clinical trials
Camrelizumab	Combined with chemotherapy (oxaliplatin, paclitaxel)	HER-2 negative, unresectable GC with programmed death ligand 1 + combined positive score ≥ 1, MSI-H/dMMR, or EBV (+) GC	Completed	NCT04694183
Sintilimab	Combined with Apatinib and chemotherapy (S-1 fluoropyrimidine, nab-paclitaxel)	Unresectable patients with stage IV GC	II	NCT04267549
Nivolumab/Pembrolizumab/Atezolizumab	Combined with chemotherapy (cyclophosphamide and fludarabine)	Advanced relapse or progression of solid tumor	I	NCT03841110
Tislelizumab	Combined with Apatinib and chemotherapy (oxaliplatin and capecitabine)	Advanced HER-2-negative GC/GEJ with signet ring cell carcinoma or peritoneal metastasis and poor prognosis	Completed	NCT06238752
Pembrolizumab	HER-2 negative group: Combined with capecitabine/oxaliplatin. HER-2 positive group: Combined with Trastuzumab and capecitabine/cisplatin	EBV negative advanced GC/GEJ and microsatellite-stable (or mismatch repair-proficient) GC	II	NCT04249739
Margetuximab/Retifanlimab/Tebotelimab	Combined with chemotherapy, chemotherapy: Capecitabine and oxaliplatin or mFOLFOX6	HER-2-positive GC/GEJ	III	NCT04082364
Camrelizumab	Combined with FOLFOX	Advanced GC/GEJ	II	NCT03939962
Atezolizumab	Combined with docetaxel, oxaliplatin, and capecitabine	Resectable GC/GEJ	II	NCT03448835
Pembrolizumab	Combined with mFOLFOX6	Potentially resectable locally advanced GC/GEJ (T1N1-3M0 or T2-3NanyM0)	II	NCT03488667
Atezolizumab	Combined with chemotherapy (docetaxel, oxaliplatin, leucovorin calcium, fluorouracil, capecitabine)	Previously untreated localized GC/GEJ with MSI-H/dMMR	II	NCT05836584
Avelumab	Combined with peri-operative FLOT	Stage Ib (T1N1 only)-IIIC GC	III	NCT03979131
Avelumab	Combined with FLOT	Resectable GC/GEJ	II	NCT03399071
Atezolizumab	Combined with FLOT	Locally advanced, resectable GC/GEJ without distant metastases	II/III	NCT03421288

EBV: Epstein-Barr virus; FLOT: Docetaxel, oxaliplatin, fluorouracil/Leucovorin; FOLFOX: Folinic acid, fluorouracil, and oxaliplatin; GC: Gastric cancer; GC/GEJ: Gastric cancer/gastroesophageal junction; HER-2: Human epidermal growth factor receptor 2; MSI-H/dMMR: Microsatellite instability-high/DNA mismatch repair.

Clinical trials of immunotherapy combined with chemotherapy for GC have demonstrated promising therapeutic effects. However, some clinical trials of combined therapy did not compare with chemotherapy or immunotherapy alone. On the other hand, drug toxicity and side effects of the combined therapy will increase. The efficacy and toxicity of drugs are influenced by the stage of GC, drug dosage and treatment duration, as well as individual differences among patients. The therapeutic effect of combined therapy is also influenced by the tumor heterogeneity, the diversity of tumor escape mechanisms, and the TME. The specific combination regimens, the type of GC which benefits, and the timing of combination therapy require further research.

The traditional view is that chemotherapy suppresses the human immune system, including suppressing tumor-killing T lymphocytes, promoting immune tolerance and inhibition, and other effects. However, in recent years, researchers have found that chemotherapy has a bidirectional regulatory effect on the body's immune microenvironment and can activate endogenous anti-tumor immune responses. Chemotherapy can promote tumor cells to release the immunogenic substances and enhance the sensitivity of immune cells. These effects are mediated by different types of chemotherapy drugs. Furthermore, studies have shown that chemotherapy drugs can induce tumor cells to express PD-L1. For example, 5-fluorouracil (5-FU) can enhance the expression of PD-L1 in GC cells. This indicates that chemotherapy can act in synergy with ICIs, and at the same time, chemotherapy can also enhance the immune response of ICIs, which provides a basis for clinical combined treatment[65]. Whether ICIs produce similar synergistic effects with different chemotherapy regimens, benefiting subgroups of patients, and the timing of combined treatment still require further in-depth research. It is also necessary to further study the impact of chemotherapy on the TME. Therefore, tumor biopsy before and during clinical trial treatment is of vital importance, as it can better reflect the changes in tumor immune status than peripheral blood markers[66].

Current research indicates that combination therapy shows great therapeutic potential and is worthy of further exploration in the future. Future research should focus on aspects such as the timing, method, and dosage of administration to achieve the best therapeutic effect.

ICIs combined with targeted therapy

Targeted therapy is a treatment aim at identified oncogenic sites at the cellular and molecular level. These sites can be anywhere in the tumor cell, they can be protein molecules on the surface of the tumor cell, or genetic material in the tumor cell. With the development of genetic engineering technology and molecular cell biology, research on the gene sequence and molecular mechanism of GC has become more in-depth and detailed, and significant breakthroughs have been made in the targeted therapy of GC[67,68]. The molecules with significant effects in GC are vascular endothelial growth factor (VEGF) and epidermal growth factor receptor (EGFR)[69].

Anti-EGFR agents include Trastuzumab, Cetuximab, and Gefitinib. Trastuzumab was the first human EGFR 2 (HER-2) mAb used in the clinic[70]. HER-2 encodes a tyrosine kinase receptor and could regulates cell growth, survival, and differentiation[71]. It is a member of the EGFR family and is closely related to human tumors, and advanced GC show high expression of EGFR. Approximately 15% to 20% of GC/GEJ patients are HER-2 positive. The signal transduction pathways mediated by HER-2 protein mainly include PI3K, AKT, RAS, and RAF, stimulating intracellular signal transduction and promoting cancer progression (Figure 2)[72,73]. HER-2-positive GC is often more aggressive and may have a poorer prognosis. Currently, the therapy of Trastuzumab combine with chemotherapy have been approved as the first-line treatment in HER-2-positive unresectable or metastatic GC[74].

Figure 2 The mechanism of epidermal growth factor receptor/ vascular endothelial growth factor receptor inhibitors. The activation of epidermal growth factor receptor (EGFR) in tumor cells triggers the phosphorylation of tyrosine residues within the receptor kinase domain and activates the Ras/Raf/mitogen-activated protein kinases (MAPK) or phosphatidylinositol 3-kinase/protein kinase B/mechanistic target of rapamycin pathways, leading to angiogenesis, cell proliferation, growth, and metastasis. The activation of vascular endothelial growth factor receptor (VEGFR) in endothelial cells can also inhibit tumor growth through the Ras/Raf/MAPK pathway. Anti-EGFR/anti-VEGFR can block these signaling pathways, preventing angiogenesis and inhibiting tumor growth. AKT: Protein kinase B; EGF: Epidermal growth factor; EGFR: Epidermal growth factor receptor; GC: Gastric cancer; M-TOR: Mechanistic target of rapamycin; PI3K: Phosphatidylinositol 3-kinase; VEGF: Vascular endothelial growth factor; VEGFR: Vascular endothelial growth factor receptor.

Studies have shown that targeting HER-2 can affect immune checkpoints in the TME. The targeted therapy of HER-2 can upregulate the expression of immune checkpoints in the TME, enhance the ADCC process, and improve the efficacy of immunotherapy[75,76]. In addition, ICIs can reduce the resistance to HER-2 targeted therapy. Therefore, when combined with ICI therapy, they can exert a synergistic effect and enhance the anti-tumor effect. ICIs combined with targeted therapy have shown good efficacy, synergistically enhancing the anti-tumor effect with good safety.

Anti-VEGF drugs are targeted drugs which mainly include Apatinib, Bevacizumab and Ramucirumab. Apatinib is the first small molecule anti-angiogenic targeted drug. And it showed safe and effective in advanced GC worldwide, and significantly extends survival after standard chemotherapy fails in advanced GC. However, Bevacizumab did not significantly prolong OS in GC patients, but significantly prolonged PFS[77]. The results showed that Bevacizumab cannot be used as a routine treatment option in advanced GC. In the phase III study, RAINBOW-Asia, paclitaxel plus Ramucirumab compared with paclitaxel achieved a median PFS of 4.14 months vs 3.15 months, and the ORR rate increased by 6% (26.5% vs 20.5%)[78]. On March 18, 2022, it was approved for second-line treatment of advanced GC/GEJ. The mechanisms of anti-VEGF drugs require further examination, and their effect on the TME has not been clearly defined. Further research on the anti-tumor mechanism and exploration of combination therapy is needed.

The combination of ICIs with targeted therapy has shown promising results. For patients who are positive for both specific targets and immune checkpoints, the combination of ICIs and targeted therapy is particularly effective. Targeted therapy enhances the tumor's sensitivity to ICI treatment. Studies have shown that targeted drugs can activate immune effector cells to release IFN-γ, increase the PD-L1 expression. Therefore, the combination of these two therapies can synergistically enhance the body's anti-tumor effects, with good safety profiles.

ICIs and targeted therapy have become important clinical treatment strategies for GC. Researchers have attempted various different combination treatment strategies for GC (Table 4)[79-92]. Combination regimens have shown good treatment effects in some refractory GC patients[93]. We explored the mechanism by which targeted and anti-PD-1 therapies produce synergistic anti-tumor activity. Although the long-term efficacy of some trials is still under continuous observation, ICIs combined with targeted therapy has shown good treatment effects in the short term. In particular, this combination seems to be more effective in patients who are double positive for the corresponding targets and immune checkpoints. The combined application of the two can synergistically enhance the body's anti-tumor effect and has good safety. The mechanism of the synergistic action between targeted drugs and ICIs, and how to select the combination therapy to achieve the best anti-GC effect still requires further study.

Table 4 Clinical trials of immune checkpoint inhibitors combined with targeted therapy for gastric cancer.

Immune checkpoint inhibitor	Target drug	Targets	Type of GC	Phase	Ref.
Pembrolizumab	Trastuzumab	HER-2	Advanced unresectable metastatic HER-2-positive GC	III	Lee et al[79]
Pembrolizumab	Trastuzumab	HER-2	Metastatic HER-2-positive GC	II	Janjigian et al[80]
Pembrolizumab	Margetuximab	HER-2	Locally advanced unresectable HER-2-positive metastatic GC, programmed death ligand 1 unselected, which had progressed after treatment with Trastuzumab combined with chemotherapy	I/II	Catenacci et al[81]
SHR-1210	Apatinib	VEGFR-2	GC/GEJ	II	Xu et al[82]
Pembrolizumab	Lenvatinib	VEGFR	Metastatic or recurrent GC/GEJ	II	Kawazoe et al[83]
Pembrolizumab	Lenvatinib	VEGFR	Advanced metastatic and/or unresectable GC which failed systemic therapy	II	Taylor et al[84]
Nivolumab	Regorafenib	VEGFR	Refractory advanced GC	III	Lam et al[85]
Nivolumab	Regorafenib	VEGFR	Unresectable recurrent GC and refractory or intolerant to chemotherapy	I	Fukuoka et al[86]
Nivolumab	Anlotinib	VEGFR	Unresectable or metastatic GC patients who failed standard therapy	II	Wu et al[87]
Pembrolizumab	Cabozantinib	VEGFR	Refractory metastatic GC	II	Dayyani et al[88]
Pembrolizumab	Ramucirumab	VEGFR-2	Previously treated advanced GC	I	Herbst et al[89]
Nivolumab	Relatlimab	Lymphocyte activation gene-3	Unresectable stage II/stage III GC	II	Kelly et al[90]
Nivolumab	Mogamulizumab	C-C chemokine receptor 4	Unable to have standard operation, refused standard preoperative chemotherapy, electrocorticography performance status 0 or 1	I	Jinushi et al[91]
Nivolumab	Bemarituzumab	FGFR	Previously untreated advanced GC with FGFR2b overexpression	Ib/II	Wainberg et al[92]

FGFR: Fibroblast growth factor receptor; GC: Gastric cancer; GC/GEJ: Gastric cancer/gastroesophageal junction; HER-2: Human epidermal growth factor receptor 2; VEGFR: Vascular endothelial growth factor receptor.

ICIs combined with Nano-antibodies

Most cancer patients benefit from these antibodies, but some patients show no response. To enhance the effectiveness of immunotherapies, including ICIs, researchers are attempting to miniaturize antibodies to reduce their immunogenicity and increase their permeability. Nanobodies are a special type of antibody found in camelid animals such as camels and llamas. These immunoglobulins consist solely of heavy chains and lack light chains, with a molecular weight that is only 10% of traditional antibodies. Therefore, they are also known as variable domains of heavy-chain antibodies. As the naturally occurring smallest functional fragment of an immunoglobulin, nanobodies have unique advantages in terms of molecular structure and physicochemical properties. They retain the full antigen-binding capacity of traditional heavy-chain antibodies, exhibiting high affinity and the ability to bind multiple epitopes. They have low immunogenicity, high stability, strong solubility, and relatively low production costs, making them widely used in biochemical mechanism research, structural biology, and the diagnosis and treatment of diseases such as cancer.

Caplacizumab, produced by Belgian company Ablynx, is the world's first nanobody drug to be marketed globally and has been designated by the FDA as a standard treatment for acquired thrombotic thrombocytopenic purpura. KN035, a nanobody developed by China's ConjuChem, is the fusion of a PD-L1 domain antibody with the Fc domain of a conventional antibody, making it the first subcutaneously administered antibody targeting PD-L1 worldwide. The Envafolimab has acceptable safety and effective in advanced microsatellite instability-high/DNA mismatch repair (MSI-H/dMMR) solid tumors which have previously received treatment. BT-100 is another nanobody with dual specificity for KRAS and signal transducer and activator of transcription 3 (STAT3). It inhibits the GTPase activity of mutated KRAS, reducing the production of downstream P-ERK and suppressing cancer cell proliferation. These mechanisms collectively result in the inhibition of human cancer tumor growth in vivo. Upon binding to STAT3, SBT-100 significantly reduces the levels of VEGF and PD-L1 transcribed by STAT3. Additionally, SBT-100 inhibits Th1 and Th17 autoimmune cells and key inflammatory cytokines such as IL-17, IFN-γ, granulocyte-macrophage colony-stimulating factor, and IL-1-α. These cytokines, along with IL-6, play crucial roles in the TME, contributing to suppression of the host's immune response against tumors. SBT-100 is a novel cell-penetrating nanobody and, as a prototype drug, it first demonstrated that targeting intracellular proteins for therapeutic effects is both feasible and safe[94].

The occurrence and development of GC is a complex dynamic process. Constructing multi-target nanocomposite antibodies or combining multiple targets for drug therapy represents a new direction for future GC treatment research[95]. However, currently, there is a lack of studies on the combination of ICIs and nanobodies in GC therapy, although the therapeutic advantages of nanobodies warrant further investigation. With in-depth research into biochemistry, human genomics, and the exploration and development of nanobodies, more efficient and specific nanobodies will be developed. This will provide a new era of targeted nanobody therapy for GC. As technology continues to advance, the nanobody platform is expected to bring significant clinical benefits to an increasing number of patients.

DISCUSSION

GC has a complex TME, providing multiple potential targets for immunotherapy. The TME of GC is mainly composed of immune cells, cytokines and extracellular matrix (ECM). The ECM is mainly composed of collagen, elastin and hyaluronic acid. It interacts with cells and regulates various functions, including cell differentiation, proliferation and migration. The ECM not only provides dynamic tissue integrity, but also participates in and drives many biological reactions as a signaling molecule. Its dysregulation is the direct or indirect cause of most chronic diseases. The ECM in tumor tissues is called "Oncomatrix", and its characteristics promote the migration, adhesion and proliferation of tumor cells[96]. The remodeling of Oncomatrix directly affects the invasiveness of tumors and the therapeutic effect. Tumor cells stimulate fibroblasts to express type I and type III collagen as well as ECM-modified enzymes, such as lysyl oxidase. This change leads to an increase in the density of tumor ECM, thereby affecting cell migration and tumor progression[97]. The high density of Oncomatrix reduces the drug's diffusion from the blood and decreases the tumor's sensitivity to chemotherapy. Maintaining the homeostasis of ECM may be a new approach in cancer prevention and can also serve as a predictive indicator for the therapeutic effect of tumors. Altering the characteristics of ECM through physical or chemical means can provide new strategies for cancer treatment.

Immune checkpoints can regulate immune responses, these proteins mainly expressed on various immune cells, especially APCs, are also present on tumor cells, while specific ligands are mainly expressed on immune cells[98]. The receptors bind with ligands to generate inhibitory signals to suppress the immune system and prevent it from overreacting. Therefore, tumor cells evade host immune surveillance and cause immune escape. ICIs activate the immune system by blocking the pathways related to suppressing T cell activity and immune responses. ICIs have developed rapidly and are a potential therapy for advanced or metastatic GC, playing an important role in various clinical trials.

However, ICI monotherapy does not provide satisfactory efficacy for many types of GC. The low immune response rate, slow process, and high selectivity for tumor types greatly limit clinical applications of ICIs in GC. Firstly, based on comprehensive genomic analysis, GC is classified into different subtypes, including microsatellite instability (MSI), chromosomal instability, EBV-positive, and genomic stability tumors[99]. The high methylation of the CpG island in the MLH1 gene promoter region is the primary cause of MSI-H/dMMR GC. This kind of GC is characterized by genomic instability and a high mutation burden. It often results in multiple genetic mutations that affect the cell cycle, DNA damage response, and the formation of various signaling pathways, such as the Wnt/β-catenin signaling pathway. These changes ultimately contribute to the development and progression of the tumor[100]. MSI-H/dMMR GC shows strong immunogenicity due to its high mutation burden and abundant tumor-infiltrating lymphocytes. PD-L1 is highly expressed in MSI-H/dMMR GC, providing a therapeutic target for ICIs. The effect of chemotherapy on MSI-H/dMMR GC is limited, especially 5-FU and cisplatin[101]. ICIs (such as PD-1/PD-L1 antibodies) have shown significant efficacy in MSI-H/dMMR GC, but some patients still have intrinsic resistance. MSI-H/dMMR GC has a special molecular type, and immunotherapy has shown promise in metastatic MSI-H/dMMR GC Therefore, detecting MSI or mismatch repair (MMR) status can be used as standard practice to guide GC treatment. Secondly, these limitations may be related to the complexity of drug interactions in the clinical environment. Patients may take other drugs simultaneously in their daily lives for complications or other diseases. Researchers have found that Lapatinib can be weakened by the proton pump inhibitors in the treatment of advanced GC. Thirdly, the high resistance of most GC patients to immunotherapy may also hinder the therapeutic effect of ICIs. Resistance mechanisms involve factors related to tumor cells, the TME, and the host. Resistance mechanisms are extremely complex and can exist simultaneously or consecutively. Due to the complexity of resistance mechanisms and the TME, combination strategies may help to overcome ICI resistance. Combination therapy addresses the reactivity issue of ICI monotherapy and enhances treatment sensitivity.

ICI combination therapeutic strategies include chemotherapy, targeted therapy, radiotherapy, immune adjuvant therapy and dual immune therapy. Each combination has obvious advantages. In this minireview, we have focused on therapeutic strategies combining ICIs with chemotherapy and targeted therapy. Due to destruction of the TME by chemotherapeutic drugs, systemic toxicity, multi-drug resistance (MDR), and immunosuppression occur after chemotherapy, which seriously affect the prognosis of patients. In the later stage of chemotherapy, tumors adaptively produce PD-L1 to counteract PD-1, creating an immunosuppressive position and evading immune surveillance. This intensifies immunity TME and weakens the anti-cancer immune response. The advantages and disadvantages of chemotherapy and ICIs seem to complement each other to some extent. ICIs have the potential to reduce systemic toxicity by reducing the dose of chemotherapeutic drugs and overcome the MDR of chemotherapy. ICIs can reverse tumor immunosuppression induced by chemotherapy, and the ICD caused by chemotherapy may accelerate the immune response process, thereby providing long-lasting immunity for the monitoring and eradication of metastatic tumors[102]. Chemical immunotherapy has made significant progress in promoting antigen presentation and enhancing immune responses. Cancer chemotherapy and immunotherapy can not only improve the advantages of both but also address their shortcomings, offering great potential for cancer treatment.

How to increase the treatment effect and reduce the side effects of treatment with ICIs combined with chemotherapy, such as the administration method, dosage, sequence, and combination of drug types, still require further exploration[103]. Different chemotherapy drugs have different anti-tumor mechanisms and cause different immune responses[104]. Therefore, evaluating the levels of immune checkpoint expression in GC to detect immune cell infiltration, cytokines, and tumor mutation burden in the TME, and choosing appropriate combination strategies based on the mechanisms of action of ICIs and chemotherapy drugs may lead to better therapeutic effects. However, in current clinical studies, there is still a lack of research on the therapeutic effects of different combination strategies[102,105].

Few clinical trials have compared the effectiveness of different combinations of chemotherapy drugs and ICIs. In most clinical trials, the administration methods and sequences are relatively simple, with chemotherapy and ICIs often administered simultaneously. However, in several animal experiments on combination therapies, different administration sequences of chemotherapy drugs and ICIs showed distinct therapeutic effects. In a study on the efficacy of ICIs combined with chemotherapy to treat colorectal cancer, intraperitoneal injection of cyclophosphamide followed by the administration of anti-CTLA-4 antibodies led to significant tumor regression. However, when the administration sequence was reversed, it caused apoptosis of tumor-specific CD8+ T cells. Chemotherapy before ICI treatment demonstrated a more effective anti-tumor effect than chemotherapy after ICI treatment[106].

The different doses of ICIs combined with chemotherapy may lead to different anti-tumor effects. Studies have shown that low doses of cisplatin and oxaliplatin can increase CD4+ and CD8+ T cells in the circulation, and reduce CD4+ and CD25+ Tregs in the TME to enhance anti-tumor effects[107]. However, high-dose regimens can suppress lymphocytes and even completely eliminate hematopoietic cells, affecting the anti-tumor effect[108,109].

In addition, when ICIs are administered in sequence with chemotherapy, the duration of the dosing interval also affects the therapeutic outcome. The content of Th1 subsets and PD-1 + CD8+ T cells was significantly higher in the longer dosing interval group. Blood tests before immunotherapy and after chemotherapy showed that the absolute neutrophil count and neutrophil-to-lymphocyte ratio were significantly higher in the short rest period group[110]. However, studies on the impact of drug administration sequence and intervals on therapeutic effects are rare, and conclusions of the trials still require further investigation. Nevertheless, this research provides a brand-new direction for the application of combination therapy.

ICIs combined with chemotherapy have demonstrated better anti-tumor effects. The mechanism by which the combination therapy synergistically combats tumors is worth further exploration in order to maximize therapeutic efficacy and increase the treatment response rate. ICIs combined with targeted therapy have produced favorable therapeutic effects in many types of cancer, and the combination therapy seems to have a role in overcoming drug resistance. Targeted drugs have cytotoxicity, which can prevent the proliferation of tumor cells, and they also have immunomodulatory effects, which can establish a favorable TME for the effectiveness of immunotherapy and have great potential to enhance the efficacy of anti-PD-1 therapy. Therefore, the combined use of targeted therapy and immunotherapy may produce greater therapeutic effects than the use of either therapy alone.

Combining anti-HER-2 and anti-VEGF/VEGF receptor mAbs with ICIs represents two major strategies[111]. However, there are still many difficulties and challenges in relation to these combination regimens. Firstly, the impact of targeted drugs in combination with ICIs on the immune system of GC patients needs to be determined to enhance the anti-GC effect[112]. Therefore, a thorough study of the response of the TME to specific targeted therapies is crucial for selecting appropriate immunotherapy. Secondly, similar to the combination with chemotherapy, the timing or sequence of combined use, the optimal regimen, and the optimal dose are still unclear and require further research. Finally, during combination treatment, both the TME and the patient's condition can change. The lack of sensitive and effective predictive biomarkers for combination therapy makes it impossible to guide clinical practice to adjust the treatment plan in a timely manner, change the combination strategy, or increase or decrease the dosage. This may lead to low efficiency in clinical decision-making, and a large number of related studies are needed.

CONCLUSION

Some chemotherapy and surgical treatments remain the primary options for GC patients. With the integration of precision medicine, immunotherapy, and targeted therapy or chemotherapy, new hopes have emerged for GC treatment. Overcoming drug resistance, determining the best combination of ICIs, the mode of administration, dosage, sequence, and frequency of administration to achieve the best PFS, OS, and minimize adverse reactions are key challenges. This requires more extensive and in-depth experimental research, ultimately leading to personalized, effective, and well-tolerated treatment methods.