PD-1 and LAG-3 dual blockade: emerging mechanisms and potential therapeutic prospects in cancer

Synergistic anti-tumor mechanism underlying anti-LAG-3 and anti-PD-1

PD-1 has been a key immune checkpoint in cancer immunotherapy. PD-1 has an important role in regulating the immune response and preventing an overactive immune system by attenuating T-cell activity through interaction with the ligands, PD-L1 or PD-L2³. The underlying mechanism is integral to the maintenance of immune homeostasis. Nevertheless, malignant cells frequently utilize the PD-1 pathway to evade immune surveillance, thereby avoiding detection and elimination by the host immune system. Therefore, PD-1 inhibitors, such as nivolumab and pembrolizumab, were developed to block the related signaling pathway by tumor cells and restore the capacity of the immune system to recognize and attack tumor cells. Clinical evidence has confirmed the effectiveness of PD-1 inhibitors⁴. PD-1 checkpoint therapy has progressed significantly but still faces notable limitations. PD-1 inhibitors are not universally effective for all patients. Additionally, the occurrence of immune-related adverse events, such as rashes and endocrine disorders, remains a significant issue requiring attention and resolution.

Considering the limitations and side effects of PD-1 blockade therapy, researchers are exploring other immune checkpoints to improve therapeutic effects. LAG-3 has received considerable interest as a promising new target. LAG-3 is an inhibitory receptor primarily expressed on exhausted T cells. LAG-3 inhibits T cell activation and function by interacting with MHC class II molecules and other ligands, including galectin-3 (Gal-3), fibrinogen-like protein 1 (FGL1), and liver and lymph node sinusoidal endothelial cell C-type lectin (LSECtin)⁵. Upregulation of LAG-3 leads to T-cell exhaustion, thereby promoting tumor immune escape. LAG-3 has a critical role in T cells and is also expressed in other immune cell types, including regulatory T and natural killer cells⁶. Clinical trials have demonstrated the significant efficacy of LAG-3 blockers in isolation or in conjunction with other immune checkpoint inhibitors (ICIs), indicating the potential to improve patient prognosis and enhance the immune system response⁷.

Studies have shown that PD-1 and LAG-3 are co-expressed in the tumor immune microenvironment, jointly mediating the immune escape of cancer cells⁸. When expressed simultaneously, PD-1 and LAG-3 synergistically inhibit T cell activation signaling through distinct but complementary pathways and activate T cell exhaustion signaling pathways. T-cell exhaustion refers to the gradual decline in T cell function within the immune system, a phenomenon influenced by various factors, including persistent antigen exposure, the impact of immunosuppressive signals, the role of soluble mediators, and the expression levels of multiple transcription factors associated with T-cell exhaustion (Figure 1). Vignali and colleagues discovered that CD8⁺ T cells with dual PD-1 and LAG-3 blockade exhibited stronger tumor clearance ability and long-term survival in a melanoma mouse model compared to CD8⁺ T cell blockade with either receptor alone. These dual-blockade CD8⁺ T cells also displayed extensive T cell receptor (TCR) clonality and enrichment of effector-like and interferon-responsive genes, resulting in increased release of IFN-γ⁹. Another clinical trial (NCT03743766) revealed the complex molecular mechanisms underlying combined LAG-3 and PD-1 blockade. The study showed that combination therapy (relatlimab and nivolumab) was safe and effective, enhancing the CD8⁺ T cell receptor signaling capacity, lowering the threshold for TCR activation, and altering CD8⁺ T cell differentiation to increase cytotoxicity while retaining exhaustion characteristics¹⁰. This research demonstrated that cytotoxicity can be enhanced while preserving exhaustion features, guiding future immunotherapy strategies. Concurrently, Wherry et al. reported that during chronic viral infections and cancer, exhausted CD8⁺ T cells persistently co-express inhibitory receptors with PD-1 and LAG-3 having distinct and complementary roles in CD8⁺ T cell exhaustion. PD-1 primarily inhibits T cell proliferation, while LAG-3 regulates T cell cytotoxicity and cytokine production. Blocking PD-1 and LAG-3 reactivates T exhaustion (Tex) cells. This finding provided new insights into how LAG-3 and PD-1 promote T-cell exhaustion differently¹¹.

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Figure 1

The causes leading to T-cell exhaustion. Ⅰ. Persistent antigen exposure: persistent antigen exposure due to chronic infection or cancer. The production of type I interferon (IFN α/β) continues to increase and the high level of IFN α/β signaling affects T-cell function, promoting T-cell exhaustion. Ⅱ. Inhibitory receptors: inhibitory receptors on the surface of T cells, such as PD-1, LAG-3, and CTLA-4, bind to the corresponding ligands on the surface of the tumor, resulting in inhibition of T-cell activation, reduction of cytokine production, and weakening of T-cell function, which leads to T-cell exhaustion. Ⅲ. Soluble mediators: soluble molecules are a second class of signals that regulate T-cell exhaustion, including immunosuppressive cytokines, such as IL-10 and transforming growth factor-beta (TGF-β), and inflammatory cytokines, such as IFN-α and IL-6. Ⅳ. Transcription factors associated with T-cell exhaustion: PR domain containing 1 (PRMD1), basic leucine zipper ATF-like transcription factor (BATF), ETS variant transcription factor 7 (ETV7), and thymocyte selection-associated high mobility group box (TOX) are transcription factors, closely associated with T-cell exhaustion. Transcription factors associated with T-cell exhaustion have an important role in regulating the CD8⁺ T-cell function and exhaustion state. The expression of transcription factors is elevated in exhausted T cells.

In summary, LAG-3 and PD-1 synergistically regulate T cell exhaustion through non-redundant mechanisms, offering new strategies for clinical anti-tumor guidance (Figure 2).

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Figure 2

The impact of the PD-1 and LAG-3 pathways on a tumor cell in blocked and unblocked states. Major histocompatibility complex (MHC) molecules and PD-L1 on the surface of tumor cells and antigen-presenting cells (APCs) interact with the inhibitory receptors LAG-3 and PD-1 on T cells, suppressing T-cell activation. By activating the PD-1 and LAG-3 immunosuppressive pathways, these immune checkpoints promote the development of exhausted T cells by inhibiting proliferation, cytokine production (IFN-γ), and cytotoxicity functions. Additionally, enhancing the expression of T-cell exhaustion-associated transcription factors, such as TOX, leads to tumor immune escape. After treatment with relatlimab and nivolumab, which effectively block the activation of the PD-1 and LAG-3 signaling pathways, the combination of immunosuppressants enhances T-cell receptor (TCR) signaling in CD8⁺ T cells and alters the differentiation of CD8⁺ T cells, restoring the proliferative capacity of T cells. Inhibition of the expression of T-cell exhaustion-related transcription factors such as TOX, simultaneously increased the release of IFN-γ and enhanced anti-tumor immunity, resulting in the effective elimination of tumor cells.

Clinical advances in combination therapy for cancer

The combination of PD-1 and LAG-3 have shown promising prospects in clinical trials. Notably, Bristol Myers Squibb developed a fixed-dose combination immunotherapy with relatlimab (anti-LAG-3 antibody) and nivolumab (anti-PD-1 antibody)¹². In a phase III clinical trial (RELATIVITY-047) involving patients with advanced melanoma, this combination significantly extended progression-free survival (PFS) from a median of 4.6 months to 10.1 months compared to nivolumab alone¹³. Based on these results, the U.S. FDA approved this combination for treating advanced melanoma in March 2022, becoming the first approved LAG-3-targeted therapy.

To validate the efficacy and breadth of this dual blockade immunotherapy, the dual blockade strategy of PD-1 and LAG-3 is gradually being tested in clinical trials for other solid tumors. In a study involving 60 patients with resectable non-small cell lung cancer, 95% achieved complete surgical resection and 12-month disease-free and overall survival (OS) rates reached 93% and 100%, respectively, following combination treatment with nivolumab and relatlimab¹⁴. In patients with metastatic head and neck squamous cell carcinoma, combination treatment with eftilagimod alpha (soluble LAG-3 protein) and pembrolizumab (PD-1 antagonist) achieved an objective response rate of 29.7% with 13.5% of patients achieving complete responses and 80% of confirmed responders maintaining response after 12 months. This result was a significant improvement compared to ICIs alone¹⁵. The combination of favezelimab (LAG-3 monoclonal antibody) and pembrolizumab (PD-1 monoclonal antibody) resulted in higher response rates and prolonged survival in patients with microsatellite-stable advanced colorectal cancer with PD-L1 CPS ≥ 1¹⁶. The combination of nivolumab and relatlimab achieved an objective response rate of 50% and PFS of 27.5% in patients with microsatellite instability-high/mismatch repair deficient (MSI-H/dMMR) metastatic colorectal cancer, providing durable clinical benefits and manageable safety for colorectal cancer patients¹⁷.

Multifaceted clinical trials indicate that combining anti-PD-1 and anti-LAG-3 therapies benefit patients who do not respond to traditional immunotherapy. Scientists have broadened the scope of immunotherapy while advancing personalized and precision treatments using this approach across various cancer types (Table 1).

Challenges of combination therapy

Combining PD-1 and LAG-3 inhibitors can improve cancer patient outcomes, advance immunotherapy, and bring new hope for survival. However, this combination therapy also faces some challenges.

Exploring future research directions in combination therapy

To enhance the effectiveness of LAG-3 and PD-1 inhibition in cancer treatment, further investigation is required to elucidate the following aspects.

Discussion

The combined inhibition strategy of PD-1 and LAG-3 has shown significant potential in current cancer immunotherapy research, especially in enhancing anti-tumor immune responses and improving patient prognosis. Clinical trials have demonstrated that this combination therapy has achieved positive results in some cancer types. For example, the fixed-dose combination of relatlimab (anti-LAG-3 antibody) and nivolumab (anti-PD-1 antibody) developed by Bristol Myers Squibb significantly extended PFS from a median of 4.6 months to 10.1 months in patients with advanced melanoma. Additionally, studies for other cancer types, such as non-small cell lung cancer and metastatic head and neck squamous cell carcinoma, have also shown promising prospects. Although clinical trials have demonstrated the positive effects of this combination therapy in certain cancer types, further investigation into the underlying mechanisms and the range of indications in other tumor types is necessary. The roles of PD-1 and LAG-3 in regulating T cell exhaustion provide new therapeutic insights, but the specific signaling pathway interactions remain to be fully elucidated. Hence, forthcoming research should focus on revealing the expression patterns and functions of these two immune checkpoints in various immune cells, especially the alterations in different tumor microenvironments. Moreover, developing reliable biomarkers to predict patient response to combination therapy will facilitate personalized treatment strategies. By identifying biomarkers to evaluate efficacy and potential adverse reactions at the onset of treatment, physicians can more effectively tailor treatment plans to improve patient outcomes and quality of life. By conducting cross-tumor studies and clinical trials we can identify the potential advantages of combination therapy in different cancers, ultimately benefiting more patients. In conclusion, despite the challenges faced by combination therapy, the prospects remain promising and deserve in-depth study.

Author contributions

Conceived and designed the analysis: Jinrong Zhu, Rongxin Zhang.

Wrote the paper: Xiangyu Qiu.

Collected the data: Zhaoan Yu, Xiaoqing Lu, Xin Jin.