Immune checkpoint blockade opens a new way to cancer immunotherapy

Among the main promising systems to triggering therapeutic antitumor immunity is the blockade of immune checkpoints. Immune checkpoint pathways regulate the control and eradication of infections, malignancies, and resistance against a host of autoantigens. Initiation point of the immune response is T cells, which have a critical role in this pathway. As several immune checkpoints are initiated by ligand–receptor interactions, they can be freely blocked by antibodies or modulated by recombinant forms of ligands or receptors. Antibodies against cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) were the first immunotherapeutics that achieved the US Food and Drug Administration approval. Preliminary clinical results with the blockers of additional immune checkpoint proteins, such as programmed cell death protein 1 (PD-1) indicate extensive and different chances to boost antitumor immunity with the objective of conferring permanent clinical effects. This study provides an overview of the immune checkpoint pathways, including CTLA-4, PD-1, lymphocyte activation gene 3, T-cell immunoglobulin and mucin domain 3, B7-H3, and diacylglycerol kinase α and implications of their inhibition in the cancer therapy.     

Development of Search Strategy for Peptide Inhibitors of Immune Checkpoints

Current strategy for the blockade of molecules inhibiting T-cell immunity, the immune checkpoints (ICP), such as CTLA-4, PD-1, and B7-H1(PD-L1), using monoclonal antibodies (mAbs), showed significant clinical effects in cancer immunotherapy. In this kind of therapy, antibodies do not kill tumor cells directly, but block inhibitory signals for T lymphocytes, resulting in activation of the immune response cascade that eliminate malignant cells and lead to tumor degradation. However, the mAb preparations have some limitations, and the development of new low-molecular-weight antagonists (for example, peptides) is an important issue. In this study, we used peptide microarrays and phage display libraries to search for peptides that interact with the immune checkpoints. We found peptides that specifically bind CTLA-4, PD-1, B7-1, B7-2 and B7-H1(PD-L1) which play important role in the regulation of the immune responses. These synthetic peptides can be applied to the development of new immunomodulating drugs for cancer immunotherapy.     

Resistance to Checkpoint Inhibition in Cancer Immunotherapy

The interaction of the host immune system with tumor cells in the tissue microenvironment is essential in understanding tumor immunity and development of successful cancer immunotherapy. The presence of lymphocytes in tumors is highly correlated with an improved outcome. T cells have a set of cell surface receptors termed immune checkpoints that when activated suppress T cell function. Upregulation of immune checkpoint receptors such as programmed cell death 1 (PD-1) and cytotoxic T lymphocyte associated protein 4 (CTLA-4) occurs during T cell activation in an effort to prevent damage from an excessive immune response. Immune checkpoint inhibitors allow the adaptive immune system to respond to tumors more effectively. There has been clinical success in different types of cancer blocking immune checkpoint receptors such as PD-1 and CTLA. However, relapse has occurred. The innate and acquired/therapy induced resistance to treatment has been encountered. Aberrant cellular signal transduction is a major contributing factor to resistance to immunotherapy. Combination therapies with other co-inhibitory immune checkpoints such as TIM-3, LAG3 and VISTA are currently being tested to overcome resistance to cancer immunotherapy. Expression of TIM-3 has been associated with resistance to PD-1 blockade and combined blockade of TIM-3 and PD-1 has demonstrated improved responses in preclinical models. LAG3 blockade has the potential to increase the responsiveness of cytotoxic T-cells to tumors. Furthermore, tumors that were found to express VISTA had an increased rate of growth due to the T cell suppression. The growing understanding of the inhibitory immune checkpoints’ ligand biology, signaling mechanisms, and T-cell suppression in the tumor microenvironment continues to fuel preclinical and clinical advancements in design, testing, and approval of agents that block checkpoint molecules. Our review seeks to bridge fundamental regulatory mechanisms across inhibitory immune checkpoint receptors that are of great importance in resistance to cancer immunotherapy. We will summarize the biology of different checkpoint molecules, highlight the effect of individual checkpoint inhibition as anti-tumor therapies, and outline the literatures that explore mechanisms of resistance to individual checkpoint inhibition pathways.     

做客肿瘤细胞的免疫检查点分子:不在其位,也谋其政

免疫检查点是一类表达在免疫细胞表面的抑制性受体分子,对维持免疫系统的稳态发挥重要的作用。近年来,陆续发现了一些重要的免疫检查点分子,例如CTLA-4和PD-1,也能在某些类型的肿瘤细胞中表达,这些“异位”表达的检查点分子被称为“肿瘤细胞固有免疫检查点分子”。虽然目前学界对它们的认识仍非常有限,但是已有证据表明,肿瘤细胞固有免疫检查点分子在表达上存在异质性,在功能上存在多样性。特别是其通过“获得性免疫非依赖”方式调控肿瘤细胞命运现象的发现,对个体化肿瘤免疫治疗方案的设计、新型抗肿瘤策略的开发都具有潜在的意义。本文概述肿瘤细胞固有免疫检查点分子的研究历程,并重点以CTLA-4和PD-1为代表,展开对肿瘤细胞固有免疫检查点分子生物学功能和分子调控机制的论述和探讨,最后对该领域存在的科学问题和未来研究方向做一展望。本综述旨在介绍和推动“肿瘤细胞固有免疫检查点分子”领域的研究。     

Inhibitor of glutamine metabolism V9302 promotes ROS-induced autophagic degradation of B7H3 to enhance antitumor immunity

Despite the enormous successes of anti-PD-1/PD-L1 immunotherapy in multiple other cancer types, the overall response rates of breast cancer remain suboptimal. Therefore, exploring additional immune checkpoint molecules for potential cancer treatment is crucial. B7H3, a T-cell coinhibitory molecule, is specifically overexpressed in breast cancer compared with normal breast tissue and benign lesions, making it an attractive therapeutic target. However, the mechanism by which B7H3 contributes to the cancer phenotype is unclear. Here we show that the expression of B7H3 is negatively related to the number of CD8⁺ T cells in breast tumor sites. In addition, analysis of the differentially expressed B7H3 reveals that it is inversely correlated to autophagic flux both in breast cancer cell lines and clinical tumor tissues. Furthermore, block of autophagy by bafilomycin A1 (Baf A1) increases B7H3 levels and attenuates CD8⁺ T cell activation, while promotion of autophagy by V9302, a small-molecule inhibitor of glutamine metabolism, decreases B7H3 expression and enhances granzyme B (GzB) production of CD8⁺ T cells via regulation of reactive oxygen species (ROS) accumulation. We demonstrate that combined treatment with V9302 and anti-PD-1 monoclonal antibody (mAb) enhances antitumor immunity in syngeneic mouse models. Collectively, our findings unveil the beneficial effect of V9302 in boosting antitumor immune response in breast cancer and illustrate that anti-PD-1 together with V9302 treatment may provide synergistic effects in the treatment of patients insensitive to anti-PD-1 therapy.     

The performance and perspectives of dendritic cell vaccines modified by immune checkpoint inhibitors or stimulants

Therapeutic dendritic cell (DC) vaccines stimulate the elimination of tumor cells by the immune system. However, while antigen-specific T cell responses induced by DC vaccines are commonly observed, the clinical response rate is relatively poor, necessitating vaccine optimization. There is evidence that the suppression of DC function by immune checkpoints hinders the anti-tumor immune responses mediated by DC vaccines, ultimately leading to the immune escape of the tumor cells. The use of immune checkpoint inhibitors (ICIs) and immune checkpoint activators (ICAs) has extended the immunotherapeutic range. It is known that both inhibitory and stimulatory checkpoint molecules are expressed by most DC subsets and can thus be used to manipulate the effectiveness of DC vaccines. Such manipulation has been investigated using strategies such as chemotherapy, agonistic or antagonistic antibodies, siRNA, shRNA, CRISPR-Cas9, soluble antibodies, lentiviruses, and adenoviruses to maximize the efficacy of DC vaccines. Thus, a deeper understanding of immune checkpoints may assist in the development of improved DC vaccines. Here, we review the actions of various ICIs or ICAs shown by preclinical studies, as well as their potential application in DC vaccines. New therapeutic interventional strategies for blocking and stimulating immune checkpoint molecules in DCs are also described in detail.     

SLAMF8, a potential new immune checkpoint molecule,is associated with the prognosis of colorectal cancer

Recently, immune checkpoint inhibitors (ICIs), such as programmed cell death 1 (PD-1) monoclonal antibodies (mAbs), have revolutionized the treatment of malignant tumors. Therefore, the number of studies aiming to screen and identify new immune checkpoint molecules for antitumor immunotherapy is increasing. Signaling lymphocytic activation molecule (SLAM) family members are mainly expressed by and regulate the functions of immune cells. Recent studies have shown that several SLAM family members are involved in the regulation of the tumor immune microenvironment and are promising targets for antitumor immunotherapy. Signaling lymphocytic activation molecule family member 8 (SLAMF8) is a type I cell surface glycoprotein and is encoded on chromosome 1q21. To further illustrate the clinical value of SLAMF8 in colorectal cancer (CRC), we retrospectively analyzed the relationship between SLAMF8 expression and the prognosis of CRC patients and the associations between SLAMF8 expression and the expression levels of other SLAM family members and other classic immune checkpoint molecules using The Cancer Genome Atlas (TCGA) data, RNA sequencing data, tissue immunohistochemistry staining, and systematic follow-up analysis. Here, high SLAMF8 expression was associated with poor overall survival (OS) in CRC. The mRNA expression level of SLAMF8 was positively correlated with the expression levels of multiple classic immune checkpoints and other SLAM family members. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis suggested that the pathways enriched in CRC tissues with high SLAMF8 expression were associated with the regulation of the tumor immune microenvironment.     

Blockade of the B7-H1/PD-1 pathway for cancer immunotherapy

The aim of cancer immunotherapy is to treat malignant disease by inducing or enhancing cancer specific immune responses. With the identification of tumor-associated antigens (TAAs) in the 1990s, cancer immunotherapy research largely focused on inducing immune responses against TAAs but achieved limited success. More recently, the underlying mechanisms and molecular pathways that cancers manipulate to subvert immune-mediated destruction have been identified, including a set of molecules with potent coinhibitory functions. Coinhibitory molecules are expressed on the surface of immune cells, cancer cells, and stromal cells and negatively regulate immune responses to cancer. In particular, one of these ligand-receptor coinhibitory interactions, B7-H1/PD-1, is critical for modulating immune responses to cancer. This knowledge led to the design of revolutionary new immunotherapeutics based on the manipulation of these molecular pathways. Monoclonal antibodies (mAbs) are the primary immunotherapeutic modality used to promote immune function via antagonism or agonism of inhibitory or stimulatory molecular pathways, respectively. Here, we review current knowledge on the function of the B7-H1/PD-1 pathway in mice and humans, its role in the subversion of immune responses in cancer, and clinical evidence that mAb targeting of this pathway results in profound immune anti-cancer effects.     

Identification of a highly immunogenic mouse breast cancer sub cell line, 4T1-S

Cancer vaccines serve as a promising clinical immunotherapeutic strategy that help to trigger an effective and specific antitumor immune response compared to conventional therapies. However, poor immunogenicity of tumor cells remains a major obstacle for clinical application, and developing new methods to modify the immunogenicity of tumor cells may help to improve the clinical outcome of cancer vaccines. 4T1 mouse breast cancer cell line has been known as poorly immunogenic and highly metastatic cell line. Using this model, we identified a sub cell line of 4T1—designated as 4T1-Sapporo (4T1-S)—which shows immunogenic properties when used as a vaccine against the same line. In 4T1-S-vaccinated mice, subcutaneous injection of 4T1-S resulted in an antitumor inflammatory response represented by significant enlargement of draining lymph nodes, accompanied with increased frequencies of activated CD8 T cells and a subpopulation of myeloid cells. Additionally, 4T1-S vaccine was ineffective to induce tumor rejection in nude mice, which importantly indicate that 4T1-S vaccine rely on T cell response to induce tumor rejection. Further analysis to identify mechanisms that control tumor immunogenicity in this model may help to develop new methods for improving the efficacies of clinical cancer vaccines.     

Immune Checkpoint Blockade to Improve Tumor Infiltrating Lymphocytes for Adoptive Cell Therapy

Tumor-infiltrating lymphocytes (TIL) has been associated with improved survival in cancer patients. Within the tumor microenvironment, regulatory cells and expression of co-inhibitory immune checkpoint molecules can lead to the inactivation of TIL. Hence, there is a need to develop strategies that disrupt these negative regulators to achieve robust anti-tumor immune responses. We evaluated the blockade of immune checkpoints and their effect on T cell infiltration and function. We examined the ability of TIL to induce tumor-specific immune responses in vitro and in vivo. TIL isolated from tumor bearing mice were tumor-specific and expressed co-inhibitory immune checkpoint molecules. Administration of monoclonal antibodies against immune checkpoints led to a significant delay in tumor growth. However, anti-PD-L1 antibody treated mice had a significant increase in T cell infiltration and IFN-γ production compared to other groups. Adoptive transfer of in vitro expanded TIL from tumors of anti-PD-L1 antibody treated mice led to a significant delay in tumor growth. Blockade of co-inhibitory immune checkpoints could be an effective strategy to improve TIL infiltration and function.     

Cancer,aging and immunotherapy: lessons learned from animal models

Aging of the immune system is associated with a dramatic reduction in responsiveness as well as functional dysregulation. This deterioration of immune function with advancing age is associated with an increased incidence of cancer. Although there is a plethora of reports evaluating the effect of immunotherapy in stimulating antitumor immune responses, the majority of these studies do not pay attention to the effect aging has on the immune system. Studies from our group and others indicate that immunotherapies could be effective in the young, are not necessarily effective in the old. To optimally stimulate an antitumor immune response in the old, it is necessary to (1) identify and understand the intrinsic defects of the old immune system and (2) use relevant models that closely reflect those of cancer patients, where self-tolerance and aging are present simultaneously. The present review summarizes some defects found in the old immune system affecting the activation of antitumor immune responses, the strategies used to activate stronger antitumor immune response in the old and the use of a tolerant animal tumor model to target a self-tumor antigen for the optimization of immunotherapeutic interventions in the old.     

Mutations and regulatory anomalies effecting tumor cell immune functions

The immune system is capable of interacting with tumor cells in such a way as to lead to tumor cell death, and this knowledge has inspired therapies to manipulate patient immune systems to eradicate cancer. However, tumor cells are able to mitigate the antitumor immune response, a fact that has rarely been addressed in the design of immunotherapies. There are many different tumor cell immune functions that play a role in mitigating the antitumor immune response. In some cases, these functions appear to be intimately associated with the tumor cell abnormalities that lead to loss of growth control, such as the cases where classical tumor suppressor proteins regulate tumor cell immune function genes. In other cases, tumor cell mutations appear to affect only the antitumor response, such as tumor cell mutations that eliminate MHC class I expression. Here I review the bases for tumor cell immune functions, noting in particular where tumor cell mutations, the gold standard for identifying a tumor-specific function, are known to be responsible for the tumor cell immune function. This review also discusses other known regulatory anomalies, in the absence of a known mutation, that are apparently important for tumor development and that regulate tumor cell immune functions. Surprisingly, in many cases where the tumor cell immune function is well understood in terms of its effect on the antitumor immune response, the tumor abnormality underlying the tumor cell immune function is completely uncharacterized.     

Dendritic cells and cytokines in immune rejection of cancer

Dendritic cells (DCs) play a crucial role in linking innate and adaptive immunity and, thus, in the generation of a protective immune response against both infectious diseases and tumors. The ability of DCs to prime and expand an immune response is regulated by signals acting through soluble mediators, mainly cytokines and chemokines. Understanding how cytokines influence DC functions and orchestrate the interactions of DCs with other immune cells is strictly instrumental to the progress in cancer immunotherapy. Herein, we will illustrate how certain cytokines and immune stimulating molecules can induce and sustain the antitumor immune response by acting on DCs. We will also discuss these cytokine-DC interactions in the light of clinical results in cancer patients.     

Antigen-specific transfer of functional programmed death ligand 1 from human APCs onto CD8+ T cells via trogocytosis

Upon specific interaction with APCs, T cells capture membrane fragments and surface molecules in a process termed trogocytosis. In this study, we demonstrate that human Ag-specific CD8(+) T cells acquire the coinhibitory molecule programmed death ligand 1 (PD-L1) from mature dendritic cells (mDC) and tumor cells in an Ag-specific manner. Immature dendritic cells were less effective in transferring surface molecules onto CD8(+) T cells than mDCs. Interestingly, trogocytosis of PD-L1 requires cell-cell contact and cannot be induced by uptake of soluble proteins obtained from mDC lysates. The transfer process is impaired by inhibition of vacuolar ATPases in T cells as well as by fixation of dendritic cells. Of importance, CD8(+) T cells that acquired PD-L1 complexes were able to induce apoptosis of neighboring programmed death 1-expressing CD8(+) T cells. In summary, our data demonstrate that human CD8(+) T cells take up functionally active PD-L1 from APCs in an Ag-specific fashion, leading to fratricide of programmed death 1-expressing, neighboring T cells. The transfer of functionally active coinhibitory molecules from APCs onto human CD8(+) T cells could have a regulatory role in immune responses.     

Inhibition of tumor growth with a vaccine based on xenogeneic homologous fibroblast growth factor receptor-1 in mice

Angiogenesis is important for the growth of solid tumors. The breaking of the immune tolerance against the molecule associated with angiogenesis should be a useful approach for cancer therapy. However, the immunity to self-molecules is difficult to elicit by a vaccine based on autologous or syngeneic molecules due to immune tolerance. Basic fibroblast growth factor (bFGF) is a specific and potent angiogenic factor implicated in tumor growth. The biological activity of bFGF is mediated through interaction with its high-affinity receptor, fibroblast growth factor receptor-1 (FGFR-1). In this study, we selected Xenopus FGFR-1 as a model antigen by the breaking of immune tolerance to explore the feasibility of cancer therapy in murine tumor models. We show here that vaccination with Xenopus FGFR-1 (pxFR1) is effective at antitumor immunity in three murine models. FGFR-1-specific autoantibodies in sera of pxFR1-immunized mice could be found in Western blotting analysis. The purified immunoglobulins were effective at the inhibition of endothelial cell proliferation in vitro and at the antitumor activity in vivo. The antitumor activity and production of FGFR-1-specific autoantibodies could be abrogated by depletion of CD4+ T lymphocytes. Histological examination revealed that the autoantibody was deposited on the endothelial cells within tumor tissues from pxFR1-immunized mice, and intratumoral angiogenesis was significantly suppressed. Furthermore, the inhibition of angiogenesis could also be found in alginate-encapsulate tumor cell assay. These observations may provide a new vaccine strategy for cancer therapy through the induction of autoimmunity against FGFR-1 associated with angiogenesis in a cross-reaction.     

Interferon-alpha and cancer: mechanisms of action and new perspectives of clinical use

Interferons-alpha (IFN-alpha) are pleiotropic cytokines belonging to type I IFNs, extensively used in the treatment of patients with some types of cancer and viral disease. IFN-alpha can affect tumor cell functions by multiple mechanisms. In addition, these cytokines can promote the differentiation and activity of host immune cells. Early studies in mouse tumor models showed the importance of host immune mechanisms in the generation of a long-lasting antitumor response after treatment of the animals with IFN-alpha/beta. Subsequently, an ensemble of studies based on the use of genetically modified tumor cells expressing specific IFN molecules provided important information on the host-mediated antitumor mechanisms induced by the local production of IFN-alpha. Of note, several studies have then underscored new immunomodulatory effects of IFN-alpha, including activities on T cells and dendritic cells, which may lead to IFN-induced antitumor immunity. In addition, recent reports on new immune correlates in cancer patients responding to IFN-alpha represent additional evidence on the importance of the interactions of IFN-alpha with the immune system for the generation of a durable antitumor response. On the whole, this knowledge suggests the advantage of using these cytokines as adjuvants of cancer vaccines and for the in vitro generation of highly active dendritic cells to be utilized for therapeutic vaccination of cancer patients.     

肾透明细胞癌中免疫检查点相关的免疫逃逸机制的研究进展

摘要：近年来,免疫治疗在晚期肾透明细胞癌的治疗中异军突起,使人们对于肾癌治疗有了全新的认识。肿瘤免疫治疗药物是通过抑制免疫检查点从而抑制肿瘤细胞免疫逃逸,使免疫细胞可以杀伤肿瘤细胞来发挥治疗作用。因此,了解肾透明细胞癌中免疫检查点相关免疫逃逸机制对于制定有效的治疗策略以及开发新的免疫治疗药物至关重要。本文对目前肾透明细胞癌中主要的免疫检查点（PD-1/PD-L1、CTLA-4、B7-H4、LAG-3、TIM-3和HLA-G）相关的免疫逃逸机制进行综述。     

Combined blockade of TIM-3 and TIM-4 augments cancer vaccine efficacy against established melanomas

Cancer vaccines have been developed to instruct the endogenous immune responses to autologous tumors and to generate durable clinical responses. However, the therapeutic benefits of cancer vaccines remain insufficient due to the multiple immunosuppressive signals delivered by tumors. Thus, to improve the clinical efficacy of cancer immunotherapy, it is important to develop new modalities to overcome immunosuppressive tumor microenvironments and elicit effective antitumor immune responses. In this study, we show that novel monoclonal antibodies (mAbs) specifically targeting either T cell immunoglobulin mucin protein-3 (TIM-3) or T cell immunoglobulin mucin protein-4 (TIM-4) enhance the therapeutic effects of vaccination against established B16 murine melanomas. This is true for vaccination with irradiated B16 melanoma cells engineered to express the flt3 ligand gene (FVAX). More importantly, combining anti-TIM-3 and anti-TIM-4 mAbs markedly increased vaccine-induced antitumor responses against established B16 melanoma. TIM-3 blockade mainly stimulated antitumor effector activities via natural killer cell-dependent mechanisms, while CD8⁺ T cells served as the main effectors induced by anti-TIM-4 mAb. Our findings reveal that therapeutic manipulation of TIM-3 and TIM-4 may provide a novel strategy for improving the clinical efficacy of cancer immunotherapy.     

Preparation of dendritic cells for cancer immunotherapy

Development of new effective method for cancer therapy is one of the most important trends in the modern medicine. Along with surgery, chemotherapy and radiotherapy, induction of an immune response against the tumor cells is a promising approach for therapy of cancer, particularly metastatic, slowly dividing tumors and cancer stem cells. Induction of the antitumor T-cell immune response involves activation of antigen-presenting cells, which can efficiently present the cancer antigens and activate T-lymphocytes. The immune response may be activated by dendritic cells (DC) loaded with tumor antigens, such as tumor-specific proteins, tumor cell lysates, apoptotic or necrotic tumor cells, as well as nucleic acids encoding tumor antigens. Regardless of the selected source of the tumor antigen, preparation of mature DC is a principal step in the development of anticancer vaccines aimed at the induction of the cytotoxic T-cell immune response. Recently, various research groups have proposed several strategies for producing mature DC, differed by the set of agents used. It has been shown that the maturation strategy influences both their phenotype and the ability to induce the immune response. In this review we have analyzed the results of studies on the various strategies of preparation of mature DCs.