Light-Controllable Binary Switch Activation of CAR T Cells

Major challenges to chimeric antigen receptor (CAR) T cell therapies include uncontrolled immune activity, off-tumor toxicities and tumor heterogeneity. To overcome these challenges, we engineered CARs directed against small molecules. By conjugating the same small molecule to distinct tumor-targeting antibodies, we show that small molecule specific-CAR T cells can be redirected to different tumor antigens. Such binary switches allow control over the degree of CAR T cell activity and enables simultaneous targeting of multiple tumor-associated antigens. We also demonstrate that ultraviolet light-sensitive caging of small molecules blocks CAR T cell activation. Exposure to ultraviolet light, uncaged small molecules and restored CAR T cell-mediated killing. Together, our data demonstrate that a light-sensitive caging system enables an additional level of control over tumor cell killing, which could improve the therapeutic index of CAR T cell therapies.     

Preclinical Evaluation of CAR T Cell Function: In Vitro and In Vivo Models

Immunotherapy using chimeric antigen receptor (CAR) T cells is a rapidly emerging modality that engineers T cells to redirect tumor-specific cytotoxicity. CAR T cells have been well characterized for their efficacy against B cell malignancies, and rigorously studied in other types of tumors. Preclinical evaluation of CAR T cell function, including direct tumor killing, cytokine production, and memory responses, is crucial to the development and optimization of CAR T cell therapies. Such comprehensive examinations are usually performed in different types of models. Model establishment should focus on key challenges in the clinical setting and the capability to generate reliable data to indicate CAR T cell therapeutic potency in the clinic. Further, modeling the interaction between CAR T cells and tumor microenvironment provides additional insight for the future endeavors to enhance efficacy, especially against solid tumors. This review will summarize both in vitro and in vivo models for CAR T cell functional evaluation, including how they have evolved with the needs of CAR T cell research, the information they can provide for preclinical assessment of CAR T cell products, and recent technology advances to test CAR T cells in more clinically relevant models.     

Genetically Modified Cellular Therapies for Malignant Gliomas

Despite extensive preclinical research on immunotherapeutic approaches, malignant glioma remains a devastating disease of the central nervous system for which standard of care treatment is still confined to resection and radiochemotherapy. For peripheral solid tumors, immune checkpoint inhibition has shown substantial clinical benefit, while promising preclinical results have yet failed to translate into clinical efficacy for brain tumor patients. With the advent of high-throughput sequencing technologies, tumor antigens and corresponding T cell receptors (TCR) and antibodies have been identified, leading to the development of chimeric antigen receptors (CAR), which are comprised of an extracellular antibody part and an intracellular T cell receptor signaling part, to genetically engineer T cells for antigen recognition. Due to efficacy in other tumor entities, a plethora of CARs has been designed and tested for glioma, with promising signs of biological activity. In this review, we describe glioma antigens that have been targeted using CAR T cells preclinically and clinically, review their drawbacks and benefits, and illustrate how the emerging field of transgenic TCR therapy can be used as a potent alternative for cell therapy of glioma overcoming antigenic limitations.     

CAR-NK Cells in the Treatment of Solid Tumors

CAR-T (chimeric antigen receptor T) cells have emerged as a milestone in the treatment of patients with refractory B-cell neoplasms. However, despite having unprecedented efficacy against hematological malignancies, the treatment is far from flawless. Its greatest drawbacks arise from a challenging and expensive production process, strict patient eligibility criteria and serious toxicity profile. One possible solution, supported by robust research, is the replacement of T lymphocytes with NK cells for CAR expression. NK cells seem to be an attractive vehicle for CAR expression as they can be derived from multiple sources and safely infused regardless of donor–patient matching, which greatly reduces the cost of the treatment. CAR-NK cells are known to be effective against hematological malignancies, and a growing number of preclinical findings indicate that they have activity against non-hematological neoplasms. Here, we present a thorough overview of the current state of knowledge regarding the use of CAR-NK cells in treating various solid tumors.     

Compact Bidirectional Promoters for Dual-Gene Expression in a Sleeping Beauty Transposon

Promoter choice is an essential consideration for transgene expression in gene therapy. The expression of multiple genes requires ribosomal entry or skip sites, or the use of multiple promoters. Promoter systems comprised of two separate, divergent promoters may significantly increase the size of genetic cassettes intended for use in gene therapy. However, an alternative approach is to use a single, compact, bidirectional promoter. We identified strong and stable bidirectional activity of the RPBSA synthetic promoter comprised of a fragment of the human Rpl13a promoter, together with additional intron/exon structures. The Rpl13a-based promoter drove long-term bidirectional activity of fluorescent proteins. Similar results were obtained for the EF1-α and LMP2/TAP1 promoters. However, in a lentiviral vector, the divergent bidirectional systems failed to produce sufficient titres to translate into an expression system for dual chimeric antigen receptor (CAR) expression. Although bidirectional promoters show excellent applicability to drive short RNA in Sleeping Beauty transposon systems, their possible use in the lentiviral applications requiring longer and more complex RNA, such as dual-CAR cassettes, is limited.     

The Effects of Chimeric Antigen Receptor (CAR) Hinge Domain Post-Translational Modifications on CAR-T Cell Activity

To improve the efficacy and safety of chimeric antigen receptor (CAR)-expressing T cell therapeutics through enhanced CAR design, we analysed CAR structural factors that affect CAR-T cell function. We studied the effects of disulphide bonding at cysteine residues and glycosylation in the HD on CAR-T function. We used first-generation CAR[V/28/28/3z] and CAR[V/8a/8a/3z], consisting of a mouse vascular endothelial growth factor receptor 2 (VEGFR2)-specific single-chain variable fragment tandemly linked to CD28- or CD8α-derived HD, transmembrane domain (TMD) and a CD3ζ-derived signal transduction domain (STD). We constructed structural variants by substituting cysteine with alanine and asparagine (putative N-linked glycosylation sites) with aspartate. CAR[V/28/28/3z] and CAR[V/8a/8a/3z] formed homodimers, the former through a single HD cysteine residue and the latter through the more TMD-proximal of the two cysteine residues. The absence of disulphide bonds did not affect membrane CAR expression but reduced antigen-specific cytokine production and cytotoxic activity. CAR[V/28/28/3z] and CAR[V/8a/8a/3z] harboured one N-linked glycosylation site, and CAR[V/8a/8a/3z] underwent considerable O-linked glycosylation at an unknown site. Thus, N-linked glycosylation of CAR[V/28/28/3z] promotes stable membrane CAR expression, while having no effect on the expression or CAR-T cell activity of CAR[V/8a/8a/3z]. Our findings demonstrate that post-translational modifications of the CAR HD influence CAR-T cell activity, establishing a basis for future CAR design.     

Modern Advances in CARs Therapy and Creating a New Approach to Future Treatment

Genetically engineered T and NK cells expressing a chimeric antigen receptor (CAR) are promising cytotoxic cells for the treatment of hematological malignancies and solid tumors. Despite the successful therapies using CAR-T cells, they have some disadvantages, such as cytokine release syndrome (CRS), neurotoxicity, or graft-versus-host-disease (GVHD). CAR-NK cells have lack or minimal cytokine release syndrome and neurotoxicity, but also multiple mechanisms of cytotoxic activity. NK cells are suitable for developing an “off the shelf” therapeutic product that causes little or no graft versus host disease (GvHD), but they are more sensitive to apoptosis and have low levels of gene expression compared to CAR-T cells. To avoid these adverse effects, further developments need to be considered to enhance the effectiveness of adoptive cellular immunotherapy. A promising approach to enhance the effectiveness of adoptive cellular immunotherapy is overcoming terminal differentiation or senescence and exhaustion of T cells. In this case, EVs derived from immune cells in combination therapy with drugs may be considered in the treatment of cancer patients, especially effector T and NK cells-derived exosomes with the cytotoxic activity of their original cells.     

Redirecting Killer T Cells through Incorporation of Azido Sugars for Tethering Ligands

The genetic expression of chimeric antigen receptors (CARs) on the surfaces of T cells enables the redirection of T cell specificity. To enhance the versatility of T cells as tumor-specific killers, we developed a nongenetic approach by which azide-containing sialic acids were metabolically incorporated into T cells to modify cellular sialyl glycans. After successful display of these moieties on the T cells, small-molecule ligands such as RGD and folate (as proof-of-concept, rather than supersized antibodies) were clicked orthogonally, leading to highly selective time- and dose-dependent cytotoxicity to integrin α_vβ₃- and folate-receptor-positive cells, respectively. This chemical approach provides a facile platform for rational design of tumor-specific cytotoxic T cells for targeted immunotherapy.     

Learning from TCR Signaling and Immunological Synapse Assembly to Build New Chimeric Antigen Receptors (CARs)

Chimeric antigen receptor (CAR) T cell immunotherapy is a revolutionary pillar in cancer treatment. Clinical experience has shown remarkable successes in the treatment of certain hematological malignancies but only limited efficacy against B cell chronic lymphocytic leukemia (CLL) and other cancer types, especially solid tumors. A wide range of engineering strategies have been employed to overcome the limitations of CAR T cell therapy. However, it has become increasingly clear that CARs have unique, unexpected features; hence, a deep understanding of how CARs signal and trigger the formation of a non-conventional immunological synapse (IS), the signaling platform required for T cell activation and execution of effector functions, would lead a shift from empirical testing to the rational design of new CAR constructs. Here, we review current knowledge of CARs, focusing on their structure, signaling and role in CAR T cell IS assembly. We, moreover, discuss the molecular features accounting for poor responses in CLL patients treated with anti-CD19 CAR T cells and propose CLL as a paradigm for diseases connected to IS dysfunctions that could significantly benefit from the development of novel CARs to generate a productive anti-tumor response.     

A transient expression vector for recombinant protein production in Chinese hamster ovary cells

An expression vector was specifically designed for use in Chinese hamster ovary (CHO) cells to enhance the level of protein production in a transient expression system. Two key components that can increase protein production transiently are the promoter used to drive recombinant gene expression and the template copy number. In this study the modified and metal‐inducible metallothionein (M2.6) promoter was shown to be superior to the human cytomegalovirus (CMV) and to the simian virus SV40 promoters. Plasmid replication was achieved using the Polyoma (Py) virus origin of replication (PyOri) and the Py Large T antigen (PyLT). An expression vector containing Py elements was shown to replicate extensively in CHO cells. The combination of the metal‐inducible M2.6 promoter and episomal replication of the expression vector, named pPyOriLT resulted in elevated levels of transgene expression following transient transfection of CHO cells Copyright © 2005 Society of Chemical Industry     

Pharmacologic Control of CAR T Cells

Chimeric antigen receptor (CAR) therapy is a promising modality for the treatment of advanced cancers that are otherwise incurable. During the last decade, different centers worldwide have tested the anti-CD19 CAR T cells and shown clinical benefits in the treatment of B cell tumors. However, despite these encouraging results, CAR treatment has also been found to lead to serious side effects and capricious response profiles in patients. In addition, the CD19 CAR success has been difficult to reproduce for other types of malignancy. The appearance of resistant tumor variants, the lack of antigen specificity, and the occurrence of severe adverse effects due to over-stimulation of the therapeutic cells have been identified as the major impediments. This has motivated a growing interest in developing strategies to overcome these hurdles through CAR control. Among them, the combination of small molecules and approved drugs with CAR T cells has been investigated. These have been exploited to induce a synergistic anti-cancer effect but also to control the presence of the CAR T cells or tune the therapeutic activity. In the present review, we discuss opportunistic and rational approaches involving drugs featuring anti-cancer efficacy and CAR-adjustable effect.     

Immune Checkpoint Blockade via PD-L1 Potentiates More CD28-Based than 4-1BB-Based Anti-Carbonic Anhydrase IX Chimeric Antigen Receptor T Cells

The complete regression of clear cell renal cell carcinoma (ccRCC) obtained pre-clinically with anti-carbonic anhydrase IX (CAIX) G36 chimeric antigen receptor (CAR) T cells in doses equivalent to ≅10⁸ CAR T cells/kg renewed the potential of this target to treat ccRCC and other tumors in hypoxia. The immune checkpoint blockade (ICB) brought durable clinical responses in advanced ccRCC and other tumors. Here, we tested CD8α/4-1BB compared to CD28-based anti-CAIX CAR peripheral blood mononuclear cells (PBMCs) releasing anti-programmed cell death ligand-1 (PD-L1) IgG4 for human ccRCC treatment in vitro and in an orthotopic NSG mice model in vivo. Using a ≅10⁷ CAR PBMCs cells/kg dose, anti-CAIX CD28 CAR T cells releasing anti-PD-L1 IgG highly decrease both tumor volume and weight in vivo, avoiding the occurrence of metastasis. This antitumoral superiority of CD28-based CAR PBMCs cells compared to 4-1BB occurred under ICB via PD-L1. Furthermore, the T cell exhaustion status in peripheral CD4 T cells, additionally to CD8, was critical for CAR T cells efficiency. The lack of hepatotoxicity and nephrotoxicity upon the administration of a 10⁷ CAR PMBCs cells/kg dose is the basis for carrying out clinical trials using anti-CAIX CD28 CAR PBMCs cells releasing anti-PD-L1 antibodies or anti-CAIX 4-1BB CAR T cells, offering exciting new prospects for the treatment of refractory ccRCC and hypoxic tumors.     

Interleukin 15 in Cell-Based Cancer Immunotherapy

Cell-based cancer immunotherapy, such as chimeric antigen receptor (CAR) engineered T and natural killer (NK) cell therapies, has become a revolutionary new pillar in cancer treatment. Interleukin 15 (IL-15), a potent immunostimulatory cytokine that potentiates T and NK cell immune responses, has demonstrated the reliability and potency to potentially improve the therapeutic efficacy of current cell therapy. Structurally similar to interleukin 2 (IL-2), IL-15 supports the persistence of CD8⁺ memory T cells while inhibiting IL-2-induced T cell death that better maintains long-term anti-tumor immunity. In this review, we describe the biology of IL-15, studies on administrating IL-15 and/or its derivatives as immunotherapeutic agents, and IL-15-armored immune cells in adoptive cell therapy. We also discuss the advantages and challenges of incorporating IL-15 in cell-based immunotherapy and provide directions for future investigation.     

丙型肝炎病毒C区和NS3区嵌合基因的克隆及表达

目的获得丙型肝炎病毒(HCV)C-NS3嵌合抗原,以提高HCV酶联免疫吸附试验诊断试剂的质量。方法应用SOE-PCR(拼接重叠延伸PCR)的方法,构建了HCV的C区和NS3区的嵌合基因,克隆于表达载体pGEX-4T3,并转化于大肠杆菌BL21,经筛选,IPTG诱导HCV的C-NS3嵌合抗原的表达。经SDS-PAGE检测表达水平,Western blot检测抗原特异性。结果SOE-PCR构建的HCV C区和NS3区嵌合基因在BL21中获得表达。经鉴定,其相对分子质量约为75000,并具有高度特异性。结论已获得HCV C-NS3嵌合抗原,为提高HCV酶联免疫吸附试验诊断试剂的质量奠定了基础。     

Cell Therapy for Colorectal Cancer: The Promise of Chimeric Antigen Receptor (CAR)-T Cells

Colorectal cancer (CRC) is a global public health problem as it is the third most prevalent and the second most lethal cancer worldwide. Major efforts are underway to understand its molecular pathways as well as to define the tumour-associated antigens (TAAs) and tumour-specific antigens (TSAs) or neoantigens, in order to develop an effective treatment. Cell therapies are currently gaining importance, and more specifically chimeric antigen receptor (CAR)-T cell therapy, in which genetically modified T cells are redirected against the tumour antigen of interest. This immunotherapy has emerged as one of the most promising advances in cancer treatment, having successfully demonstrated its efficacy in haematological malignancies. However, in solid tumours, such as colon cancer, it is proving difficult to achieve the same results due to the shortage of TSAs, on-target off-tumour effects, low CAR-T cell infiltration and the immunosuppressive microenvironment. To address these challenges in CRC, new approaches are proposed, including combined therapies, the regional administration of CAR-T cells and more complex CAR structures, among others. This review comprehensively summarises the current landscape of CAR-T cell therapy in CRC from the potential tumour targets to the preclinical studies and clinical trials, as well as the limitations and future perspectives of this novel antitumour strategy.     

TGF-β/IL-7 Chimeric Switch Receptor-Expressing CAR-T Cells Inhibit Recurrence of CD19-Positive B Cell Lymphoma

Chimeric antigen receptor (CAR)-T cells are effective in the treatment of hematologic malignancies but have shown limited efficacy against solid tumors. Here, we demonstrated an approach to inhibit recurrence of B cell lymphoma by co-expressing both a human anti-CD19-specific single-chain variable fragment (scFv) CAR (CD19 CAR) and a TGF-β/IL-7 chimeric switch receptor (tTRII-I7R) in T cells (CD19 CAR-tTRII-I7R-T cells). The tTRII-I7R was designed to convert immunosuppressive TGF-β signaling into immune-activating IL-7 signaling. The effect of TGF-β on CD19 CAR-tTRII-I7R-T cells was assessed by western blotting. Target-specific killing by CD19 CAR-tTRII-I7R-T cells was evaluated by Eu-TDA assay. Daudi tumor-bearing NSG (NOD/SCID/IL2Rγ^-/-) mice were treated with CD19 CAR-tTRII-I7R-T cells to analyze the in vivo anti-tumor effect. In vitro, CD19 CAR-tTRII-I7R-T cells had a lower level of phosphorylated SMAD2 and a higher level of target-specific cytotoxicity than controls in the presence of rhTGF-β1. In the animal model, the overall survival and recurrence-free survival of mice that received CD19 CAR-tTRII-I7R-T cells were significantly longer than in control mice. These findings strongly suggest that CD19 CAR-tTRII-I7R-T cell therapy provides a new strategy for long-lasting, TGF-β-resistant anti-tumor effects against B cell lymphoma, which may lead ultimately to increased clinical efficacy.     

The Role of Sphingolipids in Cancer Immunotherapy

Immunotherapy is now considered an innovative and strong strategy to beat metastatic, drug-resistant, or relapsing tumours. It is based on the manipulation of several mechanisms involved in the complex interplay between cancer cells and immune system that culminates in a form of immune-tolerance of tumour cells, favouring their expansion. Current immunotherapies are devoted enforcing the immune response against cancer cells and are represented by approaches employing vaccines, monoclonal antibodies, interleukins, checkpoint inhibitors, and chimeric antigen receptor (CAR)-T cells. Despite the undoubted potency of these treatments in some malignancies, many issues are being investigated to amplify the potential of application and to avoid side effects. In this review, we discuss how sphingolipids are involved in interactions between cancer cells and the immune system and how knowledge in this topic could be employed to enhance the efficacy of different immunotherapy approaches. In particular, we explore the following aspects: how sphingolipids are pivotal components of plasma membranes and could modulate the functionality of surface receptors expressed also by immune cells and thus their functionality; how sphingolipids are related to the release of bioactive mediators, sphingosine 1-phosphate, and ceramide that could significantly affect lymphocyte egress and migration toward the tumour milieu, in addition regulating key pathways needed to activate immune cells; given the renowned capability of altering sphingolipid expression and metabolism shown by cancer cells, how it is possible to employ sphingolipids as antigen targets.     

The Role of Glycosyltransferases in Colorectal Cancer

Colorectal cancer (CRC) is one of the main causes of cancer death in the world. Post-translational modifications (PTMs) have been extensively studied in malignancies due to its relevance in tumor pathogenesis and therapy. This review is focused on the dysregulation of glycosyltransferase expression in CRC and its impact in cell function and in several biological pathways associated with CRC pathogenesis, prognosis and therapeutic approaches. Glycan structures act as interface molecules between cells and their environment and in several cases facilitate molecule function. CRC tissue shows alterations in glycan structures decorating molecules, such as annexin-1, mucins, heat shock protein 90 (Hsp90), β1 integrin, carcinoembryonic antigen (CEA), epidermal growth factor receptor (EGFR), insulin-like growth factor-binding protein 3 (IGFBP3), transforming growth factor beta (TGF-β) receptors, Fas (CD95), PD-L1, decorin, sorbin and SH3 domain-containing protein 1 (SORBS1), CD147 and glycosphingolipids. All of these are described as key molecules in oncogenesis and metastasis. Therefore, glycosylation in CRC can affect cell migration, cell–cell adhesion, actin polymerization, mitosis, cell membrane repair, apoptosis, cell differentiation, stemness regulation, intestinal mucosal barrier integrity, immune system regulation, T cell polarization and gut microbiota composition; all such functions are associated with the prognosis and evolution of the disease. According to these findings, multiple strategies have been evaluated to alter oligosaccharide processing and to modify glycoconjugate structures in order to control CRC progression and prevent metastasis. Additionally, immunotherapy approaches have contemplated the use of neo-antigens, generated by altered glycosylation, as targets for tumor-specific T cells or engineered CAR (Chimeric antigen receptors) T cells.     

CAR T Cell Therapy in Hematological Malignancies: Implications of the Tumor Microenvironment and Biomarkers on Efficacy and Toxicity

Chimeric antigen receptor (CAR) T cell therapy has ushered in a new era in cancer treatment. Remarkable outcomes have been demonstrated in patients with previously untreatable relapsed/refractory hematological malignancies. However, optimizing efficacy and reducing the risk of toxicities have posed major challenges, limiting the success of this therapy. The tumor microenvironment (TME) plays an important role in CAR T cell therapy’s effectiveness and the risk of toxicities. Increasing research studies have also identified various biomarkers that can predict its effectiveness and risk of toxicities. In this review, we discuss the various aspects of the TME and biomarkers that have been implicated thus far and discuss the role of creating scoring systems that can aid in further refining clinical applications of CAR T cell therapy and establishing a safe and efficacious personalised medicine for individuals.     

Cancer-Associated Glycosphingolipids as Tumor Markers and Targets for Cancer Immunotherapy

Aberrant expression of glycosphingolipids is a hallmark of cancer cells and is associated with their malignant properties. Disialylated gangliosides GD2 and GD3 are considered as markers of neuroectoderm origin in tumors, whereas fucosyl-GM1 is expressed in very few normal tissues but overexpressed in a variety of cancers, especially in small cell lung carcinoma. These gangliosides are absent in most normal adult tissues, making them targets of interest in immuno-oncology. Passive and active immunotherapy strategies have been developed, and have shown promising results in clinical trials. In this review, we summarized the current knowledge on GD2, GD3, and fucosyl-GM1 expression in health and cancer, their biosynthesis pathways in the Golgi apparatus, and their biological roles. We described how their overexpression can affect intracellular signaling pathways, increasing the malignant phenotypes of cancer cells, including their metastatic potential and invasiveness. Finally, the different strategies used to target these tumor-associated gangliosides for immunotherapy were discussed, including the use and development of monoclonal antibodies, vaccines, immune system modulators, and immune effector-cell therapy, with a special focus on adoptive cellular therapy with T cells engineered to express chimeric antigen receptors.