Immune checkpoint inhibitors: new strategies to checkmate cancer

Immune checkpoint inhibitors (ICIs) targeting cytotoxic T lymphocyte‐associated protein‐4 (CTLA‐4) or programmed cell death protein 1 (PD‐1) receptors have demonstrated remarkable efficacy in subsets of patients with malignant disease. This emerging treatment modality holds great promise for future cancer treatment and has engaged pharmaceutical research interests in tumour immunology. While ICIs can induce rapid and durable responses in some patients, identifying predictive factors for effective clinical responses has proved challenging. This review summarizes the mechanisms of action of ICIs and outlines important preclinical work that contributed to their development. We explore clinical data that has led to disease‐specific drug licensing, and highlight key clinical trials that have revealed ICI efficacy across a range of malignancies. We describe how ICIs have been used as part of combination therapies, and explore their future prospects in this area. We conclude by discussing the incorporation of these new immunotherapeutics into precision approaches to cancer therapy.     

Combining targeted therapy with immunotherapy. Can 1 + 1 equal more than 2?

Targeted therapies have induced high response rates and improved survival in patients with cancer. However, the long-term effectiveness of targeted therapies has been limited by the development of acquired resistance in the majority of patients. On the other hand, the modern immunotherapy strategies have been associated with durable responses but in limited number of patients. Accordingly, research efforts have been focused on examining the effects of combinations of targeted therapy and immunotherapy in several different histological subtypes of cancer. There has been accumulated evidence to suggest that targeted therapy can induce immune effects in the tumor cells, the host immune system, and the tumor microenvironment. Subsequently, clinical trials have been designed to examine the efficacy of combining immune checkpoint blockade or adoptive cell transfer with tyrosine kinase inhibitors, HER family blockade, anti-angiogenic agents, histone deacetylase inhibitors, and cancer stem cell inhibitors. To date, the combination of immunotherapy with targeted therapy has demonstrated potential as a cancer treatment strategy, but further optimizations are required and caution must be taken to avoid toxicity. The current review summarizes existing evidence and provides rationale supporting the use of combined targeted and immune-therapy approaches in patients with different types of cancer.     

Targeted Therapies for Solid Tumors

The therapeutic benefits of targeted clinical interventions with increased selectivity and fewer adverse effects hold great promise in the treatment of solid malignancies, both in monotherapy and in combination. Molecular targeted therapies offer increasingly customized solutions based on the targeting of multiple specific pathways essential for cancer development and metastasis, allowing the maintenance of quality of life while efficiently attacking the tumor. To date, several monoclonal antibodies (mAbs) and small-molecule inhibitors have been approved for the treatment of colorectal, breast, head and neck, non-small cell lung and renal cell cancer. A number of additional targeted therapies are currently being investigated in ongoing clinical trials in various tumor types such as lung, gastric, cervical, uterine melanoma, and brain tumors. This article describes current and newly developed targeted therapies in solid tumors, with a special focus on tyrosine kinase inhibitors. These include mAbs and small-molecule inhibitors that aim to specifically disrupt receptor signaling pathways, which are essential for proliferation, survival and migration of tumor cells.     

Melanoma vaccines: developments over the past 10 years

Decades of preclinical evaluation and clinical trials into melanoma vaccines have yielded spectacular progress in our understanding of melanoma antigens and the immune mechanisms of tumor rejection. Key insights and the results of their clinical evaluation are reviewed in this article. Unfortunately, durable clinical benefit following vaccination remains uncommon. Two recent clinical advances that will impact on melanoma vaccine development are trials with inhibitors of CTLA-4 and oncogenic BRAF. Long-term therapeutic control of melanoma will require integration of specific active immunotherapy with these emerging successful therapies from the disparate fields of immune regulation and signal transduction.     

A new era: melanoma genetics and therapeutics

We have recently witnessed an explosion in our understanding of melanoma. Knowledge of the molecular basis of melanoma and the successes of targeted therapies have pushed melanoma care to the precipice of a new era. Identification of significant pathways and oncogenes has translated to the development of targeted therapies, some of which have produced major clinical responses. In this review, we provide an overview of selected key pathways and melanoma oncogenes as well as the targeted agents and therapeutic approaches whose successes suggest the promise of a new era in melanoma and cancer therapy. Despite these advances, the conversion of transient remissions to stable cures remains a vital challenge. Continued progress towards a better understanding about the complexity and redundancy responsible for melanoma progression may provide direction for anti-cancer drug development.     

Clinical development of phosphatidylinositol 3-kinase inhibitors for cancer treatment

The phosphatidylinositol 3-kinase (PI3K) pathway is commonly deregulated in cancer. In recent years, the results of the first phase I clinical trials with PI3K inhibitors have become available. In comparison to other targeted agents such v-raf murine sarcoma viral oncogene homolog B1 (BRAF) inhibitors in melanoma or crizotinib in anaplastic lymphoma receptor tyrosine kinase (ALK) translocated tumors, the number of objective responses to PI3K inhibitors is less dramatic. In this review we propose possible strategies to optimize the clinical development of PI3K inhibitors: by exploring the potential role of PI3K isoform-specific inhibitors in improving the therapeutic index, molecular characterization as a basis for patient selection, and the relevance of performing serial tumor biopsies to understand the associated mechanisms of drug resistance. The main focus of this review will be on PI3K isoform-specific inhibitors by describing the functions of different PI3K isoforms, the preclinical activity of selective PI3K isoform-specific inhibitors and the early clinical data of these compounds.     

HLA class I downregulation is associated with enhanced NK‐cell killing of melanoma cells with acquired drug resistance to BRAF inhibitors

The frequent development of drug resistance to targeted therapies in cancer patients has stimulated interest in strategies counteracting resistance. Combining immunotherapies with targeted therapies is one such strategy. In this context, we asked whether human NK cells can target melanoma cells that have acquired resistance to selective inhibitors targeting activating mutants of the B‐Raf kinase (BRAF inhibitors, BRAFi). We generated drug‐resistant cell variants in vitro from human BRAF‐mutant melanoma cell lines MEL‐HO, COLO‐38, SK‐MEL‐37, 1520 and from primary melanoma cells freshly isolated from two patients. All drug‐resistant cell variants remained susceptible to lysis by IL‐2‐activated NK cells; and two BRAFi‐resistant lines (BRAFi‐R) became significantly more susceptible to NK‐cell lysis than their parental lines. This was associated with significant HLA class I antigen downregulation and PD‐L1 upregulation on the drug‐resistant lines. Although blocking HLA class I enhanced the extent of lysis of both BRAFi‐R and parental cells to NK‐cell‐mediated lysis, antibody‐mediated inhibition of PD1–PD‐L1 interactions had no detectable effect. HLA class I antigen expression on BRAFi‐R melanoma variants thus appears to play a major role in their susceptibility to NK‐cell cytotoxicity. These findings suggest that NK‐cell‐based immunotherapy may be a viable approach to treat melanoma patients with acquired resistance to BRAF inhibitors.     

New perspectives in ovarian cancer treatment

Ovarian cancer (OC) is increasingly understood as a heterogeneous disease comprising distinct subtypes of different origin that vary significantly with regard to molecular biology and clinical behaviour. Despite some limited progress in its treatment over the last decade, currently there are few therapeutic options and overall survival remains poor. Increasing knowledge about the molecular biology of ovarian cancer has led to the development of targeted therapies which promise to be more effective and to provide the basis for personalized treatment. The most successful strategies so far are employing anti-angiogenics (VEGF antibodies, tyrosine kinase inhibitors and angiopoietin antagonists) and polyadenosine diphosphate-ribose polymerase (PARP) inhibitors. Other approaches target aberrant OC signalling such as the PI3K/Akt/mTOR network, the epidermal growth factor receptor, the WEE1 tyrosine kinase and the folate receptor alpha. Immunotherapy is another promising new approach against ovarian cancer. In this area, immunotherapeutic modulation by administering autologous immune cells, such as dendritic cells (DCs), to stimulate antitumour host responses is of special interest. Finally, there is now growing evidence from clinical studies showing a survival advantage for intraperitoneal (IP) chemotherapy when compared to conventional intravenous treatment in the adjuvant setting. New strategies such as pressurized IP aerosol chemotherapy might further improve the efficacy of this approach.     

Targeted Therapies in Cancer

Recent advances in understanding the biologic mechanisms underlying cancer development have driven the design of new therapeutic approaches, termed ‘targeted therapies’, that selectively interfere with molecules or pathways involved in tumor growth and progression. Inactivation of growth factors and their receptors on tumor cells as well as the inhibition of oncogenic tyrosine kinase pathways and the inhibition of molecules that control specific functions in cancer cells constitute the main rational bases of new cancer treatments tailored for individual patients. Small-molecule inhibitors and monoclonal antibodies are major components of these targeted approaches for a number of human malignancies. As the studies of the biomolecular features of cancer progress, new exciting strategies have arisen, such as targeting cancer stem cells that drive tumor relapses or the selective induction of apoptosis in malignant cells. This article primarily focuses on the biologic bases of the new cancer drugs and summarizes their mechanisms of action, the clinical evidence of their anti-cancer effectiveness as well as the rationale for their use in clinical practice.     

Kinase fusion-related thyroid carcinomas: distinct pathologic entities with evolving diagnostic implications

Activating genomic alterations in protein kinases represent a major driving force in thyroid carcinogenesis. Recently, oncogenic kinase fusions have been a central subject of pharmaceutical development, with a rapidly growing number of inhibitors validated for treating molecularly matched malignancies. Thyroid carcinomas harbor actionable kinase fusions in 10–15% of cases, occupying an increasingly recognized subpopulation of thyroid carcinomas with enhanced attention to molecular profiling. With advances in kinase-based cancer therapy, several challenges have emerged for pathologists. To interrogate an expanding list of targetable genes, the diagnostic paradigm has shifted from conventional single-gene methods toward high-throughput nucleic acid sequencing. Considering the relatively low incidence of most kinase fusions, a selective approach for molecular testing that utilizes histologic and immunohistochemical findings in triaging cases becomes essential for laboratory resource management. Moreover, kinase inhibitor resistance inevitably evolves, requiring a multimodal approach to optimal therapy, despite targeted therapies showing an enhanced, durable response. In this review, we assess the current clinicopathologic understanding and ongoing investigational topics in kinase fusion-related thyroid carcinomas.     

Targeted Therapies for Malignant Glioma

Malignant gliomas represent one of the most aggressive forms of brain cancer. Recent advances in the understanding of the deregulated molecular pathways of gliomas have brought about targeted therapies that have the ability to increase therapeutic efficacy in tumors while decreasing toxicity. Multi-targeted kinase inhibitors, novel monoclonal antibodies, and new vaccines have been developed. Standard treatments and current development of new therapies for malignant gliomas are reviewed, focusing specifically on growth factors and their receptors (e.g. epidermal growth factor receptor, vascular endothelial growth factor receptor, and platelet-derived growth factor receptor), as well as the intracellular effector molecules that are downstream of these growth factors (e.g. Ras/Raf/mitogen-activated protein kinase, phosphatidylinositol 3-kinase/AKT/mammalian target of rapamycin, and protein kinase C). The efficacies of other novel targeted inhibitors such as deacetylase inhibitors and heat shock protein 90 inhibitors in the treatment of gliomas are also discussed, as well as new combination therapies. In order for new agents to increase treatment efficacy, new targets need to be developed, drug delivery efficiency needs to be improved, and new biomarkers need to be discovered. All of these goals can be accomplished with time through innovative experimental designs.     

Targeting BRAF(V600E) in an Inducible Murine Model of Melanoma

The MAP kinase and PI3 kinase pathways have been identified as the most common pathways that mediate oncogenic transformation in melanoma, and the majority of compounds developed for melanoma treatment target one or the other of these pathways. In addition to such targeted therapies, immunotherapeutic approaches have shown promising results. A combination of these two treatment modalities could potentially result in further improvement of treatment outcome. To preclinically identify efficient treatment combinations and to optimize therapy protocols in terms of sequence and timing, mouse models will be required. We have crossed and characterized the Tyr::CreER(T2);PTEN(F-/-);BRAF(F-V600E/+) inducible melanoma model on a C57BL/6J background. Tumors from this model harbor the BRAF(V600E) mutation and are PTEN-deficient, making them highly suitable for the testing of targeted therapies. Furthermore, we crossed the model onto this specific background for use in immunotherapy studies, because most experiments in this field have been performed in C57BL/6J mice. Selective inhibition of BRAF(V600E) by PLX4720 treatment of melanoma-bearing mice resulted in a strong decrease of tumor outgrowth. Furthermore, the inducible melanomas had immune cell infiltrates similar to those found in human melanoma, and tumor-infiltrating lymphocytes could be cultured from these tumors. Our data indicate that the C57BL/6J Tyr::CreER(T2);PTEN(F-/-);BRAF(F-V600E/+) melanoma model could be used as a standard model in which targeted and immunotherapy combinations can be tested in a high-throughput manner.     

Dividing and conquering: controlling advanced melanoma by targeting oncogene-defined subsets

For decades, therapy for advanced melanoma has lagged behind most of the cancer field owing to its intrinsic resistance to conventional cytotoxic chemotherapy and limited impact of cytokine-based immunotherapy. The opportunity to develop molecularly targeted therapy emerged with the discovery of activating mutations in BRAF, a component of the long studied MAP kinase pathway. These mutations are found in approximately 50 % of patients with regionally advanced or metastatic melanoma and appear to be one of the initiating steps in the development of primary melanoma. Additional oncogenic events, particularly those that affect tumor suppressor genes, are essential for development of invasive and metastatic melanoma. Nonetheless, mutated BRAF retains its central contribution to melanoma pathophysiology even in advanced stage disease as manifested by the remarkable antitumor effects and alteration the natural history of metastatic melanoma of selective BRAF inhibitors. After initial response, resistance commonly emerges within a few months’ time and the field has focused on delineating molecular mechanisms of resistance toward the goal of improving upon the early therapeutic effects of single agent BRAF inhibition. Combination regimens are currently undergoing clinical investigation. NRAS and CKIT mutant melanoma represent the next oncogene defined melanoma subsets for which initial targeted therapy approaches are being explored, with early evidence suggesting progress with MEK and CKIT inhibitors, respectively. A considerable subset of patients have melanomas that are not defined by the presence of BRAF, NRAS, or CKIT mutations and, thus, the elucidation of the entire melanoma genome is being pursued with the hope of identifying additional therapeutic targets.     

T cell receptor therapy against melanoma—Immunotherapy for the future?

Malignant melanoma has seen monumental changes in treatment options the last decade from the very poor results of dacarbazine treatment to the modern-day use of targeted therapies and immune checkpoint inhibitors. Melanoma has a high mutational burden making it more capable of evoking immune responses than many other tumours. Even when considering double immune checkpoint blockade with anti-CTLA-4 and anti-PD-1, we still have far to go in melanoma treatment as 50% of patients with metastatic disease do not respond to current treatment. Alternative immunotherapy should therefore be considered. Since melanoma has a high mutational burden, it is considered more immunogenic than many other tumours. T cell receptor (TCR) therapy could be a possible way forward, either alone or in combination, to improve the response rates of this deadly disease. Melanoma is one of the cancers where TCR therapy has been frequently applied. However, the number of antigens targeted remains fairly limited, although advanced personalized therapies aim at also targeting private mutations. In this review, we look at possible aspects of targeting TCR therapy towards melanoma and provide an implication of its use in the future.     

Tim-3 and its role in regulating anti-tumor immunity

Immunotherapy is being increasingly recognized as a key therapeutic modality to treat cancer and represents one of the most exciting treatments for the disease. Fighting cancer with immunotherapy has revolutionized treatment for some patients and therapies targeting the immune checkpoint molecules such as CTLA-4 and PD-1 have achieved durable responses in melanoma, renal cancer, Hodgkin's diseases and lung cancer. However, the success rate of these treatments has been low and a large number of cancers, including colorectal cancer remain largely refractory to CTLA-4 and PD-1 blockade. This has provided impetus to identify other co-inhibitory receptors that could be exploited to enhance response rates of current immunotherapeutic agents and achieve responses to the cancers that are refectory to immunotherapy. Tim-3 is a co-inhibitory receptor that is expressed on IFN-g-producing T cells, FoxP3+ Treg cells and innate immune cells (macrophages and dendritic cells) where it has been shown to suppress their responses upon interaction with their ligand(s). Tim-3 has gained prominence as a potential candidate for cancer immunotherapy, where it has been shown that in vivo blockade of Tim-3 with other check-point inhibitors enhances anti-tumor immunity and suppresses tumor growth in several preclinical tumor models. This review discusses the recent findings on Tim-3, the role it plays in regulating immune responses in different cell types and the rationale for targeting Tim-3 for effective cancer immunotherapy.     

Immune checkpoints and their inhibition in cancer and infectious diseases

The development of chronic infections and cancer is facilitated by a variety of immune subversion mechanisms, such as the production of anti‐inflammatory cytokines, induction of regulatory T (Treg) cells, and expression of immune checkpoint molecules, including CTLA‐4 and PD‐1. CTLA‐4, expressed on T cells, interacts with CD80/CD86, thereby limiting T‐cell activation and leading to anergy. PD‐1 is predominantly expressed on T cells and its interaction with PD‐L1 and PD‐L2 expressed on antigen‐presenting cells (APCs) and tumors sends a negative signal to T cells, which can lead to T‐cell exhaustion. Given their role in suppressing effector T‐cell responses, immune checkpoints are being targeted for the treatment of cancer. Indeed, antibodies binding to CTLA‐4, PD‐1, or PD‐L1 have shown remarkable efficacy, especially in combination therapies, for a number of cancers and have been licensed for the treatment of melanoma, nonsmall cell lung cancer, and renal and bladder cancers. Moreover, immune checkpoint inhibitors have been shown to enhance ex vivo effector T‐cell responses from patients with chronic viral, bacterial, or parasitic infection, including HIV, tuberculosis, and malaria. Although the data from clinical trials in infectious diseases are still sparse, these inhibitors have great potential for treating chronic infections, especially when combined with therapeutic vaccines.     

Promising approaches in cancer immunotherapy

Immunotherapy is a promising field, which enhances and harnesses the powers of the host immune system against cancer and in recent years, has become a major application of the fundamental research of cancer immunology. Cancer immunotherapy is often more targeted than non-specific therapy approaches, including radiotherapy or chemotherapy, as the immune system can be trained to remember cancer cells, highlighting a durable approach that can be maintained after the treatment completion. Immunotherapy functions by directing the immune system to attack the tumour cells via targeting tumour antigens, also enhancing the existing anti-tumour immune responses. Current strategies include non-specific immunotherapy, cancer vaccines, oncolytic virus therapy, monoclonal antibodies, immune checkpoints and T cell therapy. The combination of effective approaches can increase the immunotherapy efficacy, leading to durable anti-tumour immune responses. This review will discuss the immunotherapy approaches, particularly immune checkpoints and T cell therapy, which are the most common clinical applications in cancer immunotherapy.     

EGFR assays in lung cancer

The development of small-molecule inhibitors of the epidermal growth factor receptor (EGFR) resulted in new therapeutic options for patients with advanced lung cancer. It was clear from the experience with targeted therapy for breast cancer that a new standardized assay procedure for assessing and predicting the effects of therapeutic agents must be developed. Three academic groups almost simultaneously reported the discovery of somatic mutations in the exons 18 to 21 of the tyrosine kinase (TK) domain of EGFR that correlated with a high likelihood of response to EGFR TK inhibitors. This observation revolutionized understanding of EGFR in lung carcinogenesis and resulted in numerous retrospective studies that correlated patient's response and molecular profile of the lung adenocarcinoma. The results of these studies indicate that clinical benefits from treatment with EGFR TK inhibitors are variable between the different subsets of patients. Multiple methodologic approaches were used including mutational analysis, fluorescence in situ hybridization, and immunohistochemistry. Conflicting results reflect the lack of standardization of the methodology and interpretation. Sample types, sample processing, and storage should also be taken into consideration as another potentially confounding factor. Therefore, it is important to standardize the approach and decide which assays are best to predict patient response to targeted therapies. It is also essential to determine the most cost-effective way to integrate EGFR molecular assays into clinical practice. This review will address practical aspects of each of the currently proposed assays. Difficulties in standardization of these assays in a clinical practice will be discussed.     

Targeted therapies in solid tumors: monoclonal antibodies and small molecules

The considerable progress in our understanding of the complex cellular, molecular, and genetic mechanisms underlying tumorigenesis over the last decade has fostered the development of novel and improved targeted therapies in cancer intervention. Because these therapies specifically interfere with signaling pathways essential for tumor cell proliferation, survival, and migration, they may be able to inhibit tumor growth and metastasis effectively, with fewer severe adverse events than seen with current chemotherapeutic interventions that have a narrow therapeutic index and are associated with severe toxic side effects. Monoclonal antibodies and protein tyrosine kinase inhibitors represent two classes among these promising new therapeutic interventions. This review discusses the advantages, limitations, and potential of monoclonal antibodies and protein tyrosine kinase inhibitors, and offers a perspective on the requirements and goals likely to propel the design of additional anticancer agents in the future.