Lung cancer is one of the most common malignancies, with 1.6 million new lung cancer diagnoses each year, and it is also the leading cause of death among cancer patients, with 1.4 million people dying from lung cancer each year. Treatments that target mutations in the susceptibility gene EGFR and the activation of other oncogenes have led to better outcomes for patients with more advanced NSCLC, but these therapies have not improved long-term survival, and researchers continue to search for novel treatments.
Tumorigenesis is not only due to the nature of cancer cells, but also the interaction between cancer cells and the immune system plays an important role. There are many immunotherapies used clinically for cancer treatment, such as Sipuleucel-T (a vaccine that inhibits metastasis of prostate cancer cells), Ipilimumab and interleukin-2 (IL-2) (for advanced melanoma), and interleukin-2 (IL-2) (for renal cell carcinoma).
However, due to the poor efficacy of treatment with BCG, interleukin-2(IL-2) and interferon, NSCLC was once considered to be an immunologically inactive tumor. However, with the successful development of a new generation of cancer vaccines and immunomodulators, immunotherapy for NSCLC has attracted renewed interest.
Although the promise of vaccine therapy was seen in early studies, the results of many phase III clinical trials have been unsatisfactory. Tecemotide (START study) and belagenpumatucel-L (STOP study), conducted at local technologically advanced sites, as well as studies conducted at complementary sites, suggest no therapeutic effect.
Due to genetic and epigenetic differences, the expression of cancer cell antigens differs from the expression of host cell antigens. The first step in the clearance of cancer cells by the immune system is the ability to recognize tumor cells. The T cell-mediated immune response is regulated in both directions by activating and inhibiting signals.
Negative regulatory molecules, immune checkpoint molecules, prevent the over-activation of the immune response. Checkpoint molecules (co-suppressor molecules) include cytotoxic T lymphocyte antigen-4 (CTLA-4), programmed death 1 (PD-1), TIM3, lymphocyte activation gene 3 (LAG3) and killer cell immunoglobulin receptor (KIR).
Under normal physiological conditions, these immune checkpoints protect the body from autoimmunity and inflammation. In the tumor state, these immune checkpoint proteins do not function properly, thus causing tumor tolerance and ultimately allowing tumor cells to escape from the immune system.
The use of antibodies targeting these molecules for immunomodulation is increasingly being studied, and some of them have yielded promising results. Dr. Raghav Sundar from the Institute of Oncology, National University of Singapore (NUS), et al. have reviewed co-stimulatory and co-suppressive molecules to modulate the immune response to tumors and recently developed therapeutic approaches, which were recently published in the journal Lung Cancer.
2. Co-inhibitory interventions
2.1 CTLA4
The B7 molecule, which binds to the major histocompatibility complex and APCs on antigen-presenting cells (APCs), binds to the CD28 receptor on T cells to present antigen to the T cell receptor (TCR), which in turn activates CD4 and CD8T cells.
CTLA4 binds to B7 and releases signaling molecules that allow T-cells to be reconverted to suppressor T-cells. CTLA4 also reduces IL2 production, IL2 receptor expression and directly inhibits TCR signaling molecules. activation of CTLA4 induces peripheral tolerance of antigen-specific T cells.
CTLA4 knockout mice showed significant and lethal lymphocyte infiltration, suggesting that CTLA4 plays an important role in suppressing T cell activity. expression of CTLA-4 was not only present in T lymphocytes, but researchers also detected NSCLC expression in NSCLC tumor cells in 51-87% of cases.
One of these studies showed that CTLA4 was associated with non-squamous epithelial tissue, but not with overall survival. However, in another study CTLA4 tumor expression was associated with old age and tumor hypofractionation.
The antitumor effect of CTLA4 inhibition was achieved by attenuating the inhibition of CD28/B7T cell activity. The CTLA4 inhibitor, Ipilimumab, is considered to be an effective treatment for advanced melanoma and is now being investigated for the treatment of NSCLC.
Ipilimumab is a human monoclonal antibody that binds to CTLA4, thus preventing CTLA4 from binding to the corresponding ligand and reducing the inhibition of CD28/B7T cell activity. inhibition of CTLA4 induces a rapid immune response to tumor-associated antigens.
In a randomized phase II clinical trial, patients with advanced NSCLC were randomized to 3 different treatment groups. All treatment groups received 6 rounds of paclitaxel and carboplatin chemotherapy.
Patients in group 1 (early-application group) received ipilimumab on day 1 of rounds 1-4 and were treated with placebo in the last 2 rounds of chemotherapy. Group 2 (delayed-application group) was treated with placebo during the first two rounds of chemotherapy and ipilimumab on day 1 of rounds 3-6. Group 3 (control group) received only the combination of placebo. After completing chemotherapy, patients in the first two groups continued to receive ipilimumab every 12 weeks until disease progression occurred.
Taking immune-related response criteria into account immune-related progression-free survival (irPFS) was the study’s primary study endpoint. In the delayed-application group, irPFS was 5.7 versus 4.6 months, whereas in the early-application group, irPFS did not improve. In the delayed application group, the improvement in OS was not statistically significant. Although not statistically significant, patients with squamous cell carcinoma had a longer OS.
The side effects of the therapy were rash, pruritus and diarrhea. The proportion of grade 3/4 irAE was 20% in the early-application group and 15% in the late-application group, compared with 6% in the control group. There was one death due to toxic epidermal necrolysis caused by ipilimumab.
A large phase III clinical trial in squamous carcinoma in NSCLC is ongoing, as is a study of ipilimumab in combination with EGFR and ALK tyrosine kinase inhibitors. There are also studies investigating the role of ipilimumab in small cell lung cancer.
Tremelimumab, a monoclonal antibody similar to ipilimumab, is in a phase II clinical trial in pretreated patients with advanced NSCLC. All patients received 4 rounds of platinum-based chemotherapy before being placed in two treatment arms, the Tremelimumab arm and the best maintenance therapy arm, with an ORR of 5% and no difference in PFS between the two arms.
2.2 PD-1
PD-1 receptors are expressed on CD4 and CD8 lymphocytes, Tregs, B lymphocytes and NK cells. Known ligands for PD-1 include PD-L1 (or CD274, B7-H1) and PD-L2 (CD273, B7-DC).PD-1 with PD-L1 or PD-L2 causes a decrease in cytokine production, reduced cell proliferation and cytolysis. In a variety of tumors, PD-1 expression is upregulated in tumor-infiltrating T lymphocytes (TILs), yet PD-L1 expression is also increased in many tumors.
PD-1 antagonists include PD-L1 antibodies, such as nivolumab, lambrolizumab and pidilizumab, as well as the fusion protein AMP-224.
Nivolumab is a fully human IgG4 monoclonal antibody with no detectable cytotoxicity-dependent antibody (ADCC) yet.
In phase I clinical studies in advanced solid tumors, nivolumab was administered in increasing biweekly doses for 12 rounds (2 years). In the NSCLC cohort study (n=129), the majority of patients had received multiple rounds of chemotherapy, with 55% of them receiving at least 3 regimens.
The median ORR at the median response course (74 weeks) was 17%. The median survival rate was 9.9 months, with 1- and 2-year survival rates of 42% and 24%, respectively. The median PFS was only 2.3 months.
Nivolumab was generally well tolerated, with skin toxicity (20%), gastrointestinal reactions (15%), and pulmonary symptoms (9%) being the most common adverse events. The incidence of gastrointestinal toxicity was lower with Nivolumab compared with ilipmumab. There were 2 deaths in patients who developed pneumonia.
Biomarker analysis for PD-L1 expression was performed in 63/129 patients, and tumor cells were considered PD-L1 positive if at least 5% of them showed PD-L1 expression by immunohistochemistry, with a PD-L1 positivity rate of 43%. the ORRs for PD-L1-positive and PD-L1-negative patients were 16% and 13%, respectively, indicating that long-stored tumor tissue was not suitable for detecting PD-L1 expression in the treated study group.
Enrollment in the phase III trial comparing nivolumab with paclitaxel in squamous or non-squamous cancers of NSCLC has been completed and results are forthcoming.
Lambrolizumab is a monoclonal antibody that targets PD-1 and has antitumor activity in the treatment of melanoma. A phase I clinical trial in patients with NSCLC found a median survival time of 51 weeks with Lambrolizumab and a partial efficacy rate of 25% using immune-related response criteria. Common adverse events were malaise, rash and pruritus, pneumonia in one patient, and pulmonary edema in one patient.
In the tumor marker assay study, PD-L1 expression on tumor cells obtained by immunohistochemical staining of fresh tumor tissue obtained prior to treatment was used to predict treatment response, with an ORR of 67% for PD-L1 positivity and 4% for PD-L1 negativity. Based on these results, a randomized phase II/III clinical trial comparing the efficacy of lambrolizumab and paclitaxel in PD-L1 positive patients with advanced NSCLC has been conducted.
2.3 PD-L1
Programmed death receptor ligand 1 (PD-L1, B7-H1), a ligand for PD-1, is a member of the B7 superfamily and is involved in the negative regulation of the immune response. PD-L1 is expressed by T cells, B cells, macrophages and dendritic cells, and is upregulated in many solid tumors, including NSCLC.
PD-L1 activates PD-1 on T cells due to the induction of cytokines such as IL-4, IL-10, INF-а, -B or -γ, and down-regulates T cell effector function and is one of the mechanisms by which tumor cells can invade the host immune surveillance.27-57.5% of NSCLC cells express PD-L1 and PD-L1 is present in the cell membrane and/or cell plasma. In sarcomatoid NSCLC, the percentage of PD-L1 expression is 69%, much higher than the 27% in normal NSCLC.
The prognostic impact of PD-L1 is unclear and the results are conflicting. The variation in results can be attributed to differences in the methodologies used to detect and score PD-L1 expression in different studies. It has been suggested that PD-L1 expression is associated with vascular invasion and hyperdifferentiation of tumors, but does not improve recurrence-free survival.
PD-L1 expression was associated with an increase in macrophages and dendritic cells and an increased degree of inflammatory infiltration. In contrast, PD-L1 expression was found to be negatively correlated with TILs in one study.
Histologically, PD-L1 expression was associated with squamous carcinoma, but one study showed an association with adenocarcinoma as well. One study found that PD-L1 expression was not associated with EGFR/KRAS mutations or ALK rearrangements. Considering the important role of PD-L1 in lung cancer, PD-L1 inhibition is a promising therapeutic approach. PD-L1 inhibitors currently under investigation include the monoclonal antibodies MPDL3280A, BMS-936559, MEDI4736 and MSB0010718C.
In a phase I clinical study, BMS-936559 had an ORR of 10% in solid tumors treated with NSCLC, and 18% of patients experienced at least 24 weeks of stable disease. However, the development of BMS-936559 was halted due to the high cost.
MPDL3280A is a synthetic IgG anti-PD-L1 antibody containing a modified Fc structural domain that prevents other cells from expressing PD-L1 due to antibody-dependent cell-mediated cytotoxicity (ADCC).
In the Phase I clinical study of MPDL3280A, treatment of previously treated patients with advanced NSCLC had an ORR of 24% and a 24-week PFS of 46%. The ORRs for patients with positive and negative tumor expression of PD-L1 were 100% and 15%, respectively. The ORR was 25% for former/current smokers compared to 16% for non-smokers.
MEDI4736, similar to MPDL3280A, is a synthetic Fc domain containing a tertiary mutation, thereby avoiding Fc-mediated ADCC. previous phase I clinical studies in patients with solid tumors have demonstrated no dose-limiting toxicity in terms of clinical activity and duration of disease stabilization in different tumors, including NSCLC, and no grade 3-4 treatment-related adverse events.
2.4 TIM3
Unlike other immune checkpoint molecules, TIM3 is not upregulated in all T cells upon activation, but only in CD4+ helper T cells 1 (Th1) and CD8+ cytotoxic T cells, where it participates in a synergistic suppressive effect. Upon activation by its ligand galectin-9, TIM3 suppresses the activity of effector T cells and induces peripheral tolerance.TIM3 plays a key role in T cell attrition in tumors.
In melanoma patients, TIM3 co-expression with PD-1 is detected; this co-expression indicates substantial depletion of CD8+ T cells in the tumor. In NSCLC patients, TIM3 expression was upregulated only in CD4+ and CD8+ TILs, but not in peripheral blood T cells, and TIM-3 expression in CD4+ T cells was associated with lymph node metastasis and advanced lung cancer.
A preclinical study has shown that combined inhibition of TIM3 and PD-1 is superior to inhibition alone in the treatment of selected solid tumors.
2.5 LAG3
Lymphocyte activation gene 3 (LAG3) is a costimulatory receptor expressed on activated T cells, Tregs, dendritic T cells and NK cells.LAG3 is a CD4-associated protein that binds to the type II major histocompatibility complex (MHC) and inhibits T cell proliferation, leading to tumor invasion. Upregulation of LAG3 gene expression is observed in silica-mediated lung inflammation in animal models.
A phase I clinical study of BMS-986016, a monoclonal antibody to LAG3 for the treatment of advanced solid tumors, is underway.
2.6 KIR
KIR, a regulator of the NK cell family, has recently emerged as a new target for a variety of tumor immunotherapies. Lirilumab, an antibody that binds to KIR2DL1-2 and 3 receptors, induces NK cell-mediated cell killing. increased co-expression of KIR2DL1 has been reported in NSCLC patients, which reduces NK cell function.
Two phase I clinical studies of lirilumab in combination with ipilimumab and nivolumab for the treatment of NSCLC are under investigation.
2.7BTLA and A2AR
The synergistic inhibitory receptor B and T lymphocyte attenuator (BTLA) in autoimmunity and its ligands have been investigated in relation to lymphangiogenesis-associated herpesvirus entry mediators (HVEM). a2A adenosine receptor (A2AR) is a G protein-coupled receptor that binds adenosine and has a regulatory role in the MAPK kinase pathway. There are no studies of these molecules in lung cancer.
3. Co-stimulatory interventions
3.1 OX40
OX-40 (CD134, TNFRSF4 tumor necrosis factor receptor superfamily, member 4) is a costimulatory molecule that appears in T cells activated at sites of inflammation and regulates antigen-specific T cell proliferation, survival and production of cytokines IL-2, IL-4, IL-5, and IFN-γ. The immunoreactive signaling pathway of OX40 makes it a novel target for immunotherapy.
Preclinical studies have demonstrated antitumor activity of OX40 antagonists in the treatment of melanoma, glioma, breast, colon, sarcoma, kidney, and prostate cancers.
In phase I clinical studies, patients with solid tumors were treated with three different doses of the same murine anti-human OX40 antibody in one round. Toxicity was found to be tolerable and included malaise, fever/chills, transient lymphopenia, and mild rash. 12 of the 31 patients showed tumor shrinkage, and both humoral and cellular immunity were enhanced. High levels of human anti-mouse antibodies were detected, thus necessitating the development of human OX40 antagonists.
Studies are underway for the use of OX40 antagonists in combination with radiotherapy in patients with breast and prostate cancer.
3.2 CD137
CD137 (4-1BB) is an inducible T cell surface molecule in the tumor necrosis factor (TNF) receptor superfamily. Urelumab (BMS663513), an antagonist of CD137, has been used in phase I/II clinical trials and has been found to have antitumor activity despite severe hepatotoxicity.
Nevertheless, trials with urelumab for the treatment of NSCLC have been discontinued.
3.3 GITR
GITR, a member of the TNF receptor superfamily, acts as a co-stimulatory molecule for CD4+ and CD8+ naïve T cells, causing T cell proliferation and elevating effector function.GITR is seen in Treg, effector T cells, B cells, NK cells and activated dendritic cells.TRX518, a monoclonal antibody GITR antagonist, is currently in phase I clinical studies.
3.4 CD40, CD28, CD27 and ICOS
CD40A is a member of the tumor necrosis factor superfamily, which is involved in cell differentiation, survival and apoptosis. Preclinical findings suggest that anti-CD40 antibodies may be able to inhibit tumor growth and metastasis. CD27, another T cell stimulatory receptor, also acts as an antagonist and is currently in phase I clinical studies. Inducible T-cell co-stimulator (ICOS) is a T-cell co-stimulator associated with CD28.
TGN1412, an antibody to CD28, has been discontinued due to its excessive toxicity.
4. Combination therapy
The aim of combination therapy is to target multiple tumorigenic pathways simultaneously for antitumor treatment. The first line of treatment for NSCLC is platinum. With the advent of molecularly targeted therapy for NSCLC, combinations have come into the picture.
While some combinations of targeted therapy and chemotherapy can benefit patients, some combinations can cause significant harm to patients. Some single agents are considered therapeutically effective, but in combination they do not improve patient survival, and further studies are needed to clarify these phenomena.
Phase III clinical studies of CTL4 inhibitors in combination with chemotherapeutic agents have focused on the role of ipilimumab in combination with paclitaxel and carboplatin in the treatment of squamous NSCLC. nivolumab and Ipilimumab in combination with KIR inhibitors and nivolumab in combination with multiple chemotherapeutic agents and targeted therapies have been described previously.
The combination of different immunosuppressive agents is also of interest and is already in the testing phase. CTLA4 was blocked after vaccination with secreted GM-CSF synthesized from autologous tumor cells, suggesting that antitumor immunotherapy for advanced melanoma may not be associated with significant toxicity. In a preclinical model, multiple immune checkpoint blockade using PD1 and CTLA4 antibodies resulted in enhanced T cell responsiveness and reduced T cell dysfunction.
The timing of immunotherapy in antitumor therapy remains to be investigated. Delayed application of ipilimumab was more effective than early application in a phase II clinical study, but the reasons for this are unclear.
5. Predictive Biomarkers
Predictive biomarkers are necessary because immunomodulators cause bidirectional regulation in some specific populations. Many biomarkers of CTLA4 are already in clinical use. Elevated absolute lymphocyte counts are thought to correlate with clinical efficacy. Studies have shown a positive correlation between the efficacy of ipilimumab and high levels of CD4+ T cell expression in ICOS.
HLA-DR expression was increased in CD4T cells after ipilimumab treatment. Tumor-infiltrating lymphocytes correlated with ipilimumab efficacy, and Forkhead box P3(FOXP3)-positive and indoleamine2,3-dioxygenase-positive patients responded well to CTLA4 blockade therapy.
PD-L1 expression varies across the tumor microenvironment and does not cease at a certain point in time. PD-L1 expression correlates with the presence of IFN-γ in the tumor microenvironment, which may explain why PD-L1 expression cannot be used as a predictive biomarker for nivolumab efficacy. In the field of oncology therapy, the research of predictive biomarkers is still in its infancy.
6. Assessment of tumor response to immune agents
The widely accepted method for assessing the efficacy of antitumor therapy is the RECIST standard. However, according to RECIST, a 30% or greater increase in tumor volume, even in the early stages of treatment, is considered to be disease progression. However, therapeutic response to immunotherapy is not evident until after disease progression as defined by RECIST. Despite significant tumor progression, patients continue to derive clinical benefit from immunotherapy, and sustained SD indicates maintenance of efficacy.
Based on these statements, immune-related response criteria have emerged. Immune-related progression-free survival (irPFS) can indicate maintenance of efficacy despite significant tumor enlargement. This phenomenon, called “pseudoprogression”, may be due to lymphocytic infiltration around the cancer or to a delayed immune response.
7. Conclusion
The increased understanding of tumor surveillance in the immune system has contributed to the development of new immune agents. In particular, it is now widely believed that tumors are capable of invading the immune system by dysregulating synergistic inhibition or checkpoint signaling molecules. Early research on immune checkpoint modulators such as CTLA4, PD-1 and PD-L1 inhibitors in solid tumors, such as NSCLC, is promising and will bring new light to the treatment of NSCLC.
Future challenges include the combination of different immunotherapeutic agents, or the combination of immunotherapeutic agents with cytotoxic chemotherapeutic agents, the combination of molecularly targeted therapies and radiotherapy, and the discovery of efficacy and predictive biomarkers for their application in the early stages of the disease.