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Simple Summary: The ectopic expression of the master regulator PU.1 could prove detrimental to CAR T cell functionality despite its upregulation of multiple costimulatory receptors on CAR T cells. These data proffer novel insights into CAR T cell biology and highlight the intricate quest for refining CAR T cell functionality. Chimeric antigen receptor (CAR) T cell therapy has achieved extraordinary success in eliminating B cell malignancies; however, so far, it has shown limited efficacy in the treatment of solid tumors, which is thought to be due to insufficient CAR T cell activation. We hypothesized that the transcription factor PU.1, a master regulator of innate cell functionality, may augment pro-inflammatory CAR T cell activation. T cells were engineered with a CEA-specific CAR together with the constitutive expression of PU.1. CAR-redirected T cell activation was recorded for canonical functionality in vitro under conditions of prolonged repetitive antigen exposure. Ectopic PU.1 expression in CAR T cells upregulated the costimulatory receptors CD40, CD80, CD86, and CD70, which, unexpectedly, did not augment effector functions but hampered the upregulation of 4-1BB, decreased IL-2 production, reduced CAR T cell proliferation, and impaired their cytotoxic capacities. Under "stress" conditions of repetitive engagement of cognate tumor cells, CAR T cells with ectopic PU.1 showed reduced persistence, and finally failed to control the growth of cancer cells. Mechanistically, PU.1 caused CAR T cells to secrete IFN-β, a cytokine known to promote CAR T cell attrition and apoptosis. Collectively, PU.1 can polarize the functional capacities of CAR T cells towards innate cells. [ABSTRACT FROM AUTHOR]

Chimeric antigen receptor (CAR) T cell therapy has achieved extraordinary success in eliminating B cell malignancies; however, so far, it has shown limited efficacy in the treatment of solid tumors, which is thought to be due to insufficient CAR T cell activation. We hypothesized that the transcription factor PU.1, a master regulator of innate cell functionality, may augment pro-inflammatory CAR T cell activation. T cells were engineered with a CEA-specific CAR together with the constitutive expression of PU.1. CAR-redirected T cell activation was recorded for canonical functionality in vitro under conditions of prolonged repetitive antigen exposure. Ectopic PU.1 expression in CAR T cells upregulated the costimulatory receptors CD40, CD80, CD86, and CD70, which, unexpectedly, did not augment effector functions but hampered the upregulation of 4-1BB, decreased IL-2 production, reduced CAR T cell proliferation, and impaired their cytotoxic capacities. Under “stress” conditions of repetitive engagement of cognate tumor cells, CAR T cells with ectopic PU.1 showed reduced persistence, and finally failed to control the growth of cancer cells. Mechanistically, PU.1 caused CAR T cells to secrete IFN-β, a cytokine known to promote CAR T cell attrition and apoptosis. Collectively, PU.1 can polarize the functional capacities of CAR T cells towards innate cells.

Immunotherapy has been the fifth pillar of cancer treatment in the past decade. Chimeric antigen receptor (CAR) T-cell therapy is a newly designed adoptive immunotherapy that is able to target and further eliminate cancer cells by engaging with MHC-independent tumor-antigens. CAR T-cell therapy has exhibited conspicuous clinical efficacy in hematological malignancies, but more than half of patients will relapse. Of note, the efficacy of CAR T-cell therapy has been even more disappointing in solid tumors. These challenges mainly include (1) the failures of CAR T-cells to treat highly heterogeneous solid tumors due to the difficulty in identifying unique tumor antigen targets, (2) the expression of target antigens in non-cancer cells, (3) the inability of CAR T-cells to effectively infiltrate solid tumors, (4) the short lifespan and lack of persistence of CAR T-cells, and (5) cytokine release syndrome and neurotoxicity. In combination with these characteristics, the ideal CAR T-cell therapy for solid tumors should maintain adequate T-cell response over a long term while sparing healthy tissues. This article reviewed the status, clinical application, efficacy, safety, and challenges of CAR T-cell therapies, as well as the latest progress of CAR T-cell therapies for solid tumors. In addition, the potential strategies to improve the efficacy of CAR T-cells and prevent side effects in solid tumors were also explored.

Simple Summary: Adoptive transfer of T cells targeting tumors still remains mainly ineffective in solid entities. In this review we discuss challenges related to the tumor microenvironment (TME) and promising strategies to improve tumor control by engineering the TME or the transferred T cells themselves. T cells are important players in the antitumor immune response. Over the past few years, the adoptive transfer of genetically modified, autologous T cells—specifically redirected toward the tumor by expressing either a T cell receptor (TCR) or a chimeric antigen receptor (CAR)—has been adopted for use in the clinic. At the moment, the therapeutic application of CD19- and, increasingly, BCMA-targeting-engineered CAR-T cells have been approved and have yielded partly impressive results in hematologic malignancies. However, employing transgenic T cells for the treatment of solid tumors remains more troublesome, and numerous hurdles within the highly immunosuppressive tumor microenvironment (TME) need to be overcome to achieve tumor control. In this review, we focused on the challenges that these therapies must face on three different levels: infiltrating the tumor, exerting efficient antitumor activity, and overcoming T cell exhaustion and dysfunction. We aimed to discuss different options to pave the way for potent transgenic T cell-mediated tumor rejection by engineering either the TME or the transgenic T cell itself, which responds to the environment. [ABSTRACT FROM AUTHOR]

T cells are important players in the antitumor immune response. Over the past few years, the adoptive transfer of genetically modified, autologous T cells—specifically redirected toward the tumor by expressing either a T cell receptor (TCR) or a chimeric antigen receptor (CAR)—has been adopted for use in the clinic. At the moment, the therapeutic application of CD19- and, increasingly, BCMA-targeting-engineered CAR-T cells have been approved and have yielded partly impressive results in hematologic malignancies. However, employing transgenic T cells for the treatment of solid tumors remains more troublesome, and numerous hurdles within the highly immunosuppressive tumor microenvironment (TME) need to be overcome to achieve tumor control. In this review, we focused on the challenges that these therapies must face on three different levels: infiltrating the tumor, exerting efficient antitumor activity, and overcoming T cell exhaustion and dysfunction. We aimed to discuss different options to pave the way for potent transgenic T cell-mediated tumor rejection by engineering either the TME or the transgenic T cell itself, which responds to the environment.

In the past decade, chimeric antigen receptor (CAR) T cell technology has revolutionized cancer immunotherapy. This strategy uses synthetic CARs to redirect the patient’s own immune cells to recognize specific antigens expressed on the surface of tumor cells. The unprecedented success of anti-CD19 CAR T cell therapy against B cell malignancies has resulted in its approval by the US Food and Drug Administration (FDA) in 2017. However, major scientific challenges still remain to be addressed for the broad use of CAR T cell therapy. These include severe toxicities, limited efficacy against solid tumors, and immune suppression in the hostile tumor microenvironment. Furthermore, CAR T cell therapy is a personalized medicine of which the production is time- and resource-intensive, which makes it very expensive. All these factors drive new innovations to engineer more powerful CAR T cells with improved antitumor activity, which are reviewed in this manuscript.

The dismal prognosis of pediatric and young adult patients with high-risk rhabdomyosarcoma (RMS) underscores the need for novel treatment options for this patient group. In previous studies, the tumor-associated surface antigen ERBB2 (HER2/neu) was identified as targetable in high-risk RMS. As a proof of concept, in this study, a novel treatment approach against RMS tumors using a genetically modified natural killer (NK)-92 cell line (NK-92/5.28.z) as an off-the-shelf ERBB2-chimeric antigen receptor (CAR)-engineered cell product was preclinically explored. In cytotoxicity assays, NK-92/5.28.z cells specifically recognized and efficiently eliminated RMS cell suspensions, tumor cell monolayers, and 3D tumor spheroids via the ERBB2-CAR even at effector-to-target ratios as low as 1:1. In contrast to unmodified parental NK-92 cells, which failed to lyse RMS cells, NK-92/5.28.z cells proliferated and became further activated through contact with ERBB2-positive tumor cells. Furthermore, high amounts of effector molecules, such as proinflammatory and antitumoral cytokines, were found in cocultures of NK-92/5.28.z cells with tumor cells. Taken together, our data suggest the enormous potential of this approach for improving the immunotherapy of treatment-resistant tumors, revealing the dual role of NK-92/5.28.z cells as CAR-targeted killers and modulators of endogenous adaptive immunity even in the inhibitory tumor microenvironment of high-risk RMS.

In the recent years, immunotherapy has achieved impressive results utilizing different approaches, from (chimeric antigen receptor) CAR-T cells directed against CD19 for the treatment of leukemia and lymphomas to the Nobel prize-winning strategy of check-point inhibition for the treatment of several solid tumors [...]

T cells engineered with chimeric antigen receptors (CARs) show great promise in the treatment of some cancers. Modifying T cells to express CARs generally relies on T-cell transduction using viral vectors carrying a transgene, resulting in semi-random DNA integration within the T-cell genome. While this approach has proven successful and is used in generating the Food and Drug Administration (FDA, USA) approved B-lymphocyte antigen CD19-specific CAR T cells, it is possible the transgene could integrate into a locus that would lead to malignant transformation of the engineered T cells. In addition, manufacturing viral vectors is time-consuming and expensive. One way to overcome these challenges is site-specific gene integration, which can be achieved through clustered regularly interspaced short palindromic repeat (CRISPR) mediated editing and non-viral DNA, which serves as a template for homology-directed repair (HDR). This non-viral gene editing approach provides a rapid, highly specific, and inexpensive way to engineer T cells. Here, we describe an optimized protocol for the site-specific knock-in of a large transgene in primary human T cells using non-viral double stranded DNA as a repair template. As proof-of-principle, we targeted the T-cell receptor alpha constant (TRAC) locus for insertion of a large transgene containing green fluorescence protein (GFP) and interleukin-15 (IL-15). To optimize the knock-in conditions we tested template DNA concentration, homology arm length, cell number, and knock-in efficiency over time. We then applied these established guidelines to target the TRAC or interleukin-13 (IL-13) locus for the knock-in of synthetic molecules, such as a CAR, bispecific T-cell engager (BiTE), and other transgenes. While integration efficiency depends on the targeted gene locus and selected transgene, this optimized protocol reliably generates the desired insertion at rates upwards of 20%. Thus, it should serve as a good starting point for investigators who are interested in knocking in transgenes into specific loci.

Evidences are accumulating that CD4+ T cells can physiologically mediate antigen specific target cell lysis. By circumventing major histocompatibility complex (MHC)-restrictions through an engineered chimeric antigen receptor (CAR), CD4+ T cells lyse defined target cells as efficiently as do CD8+ T cells. However, the cytolytic capacity of redirected CD4+CD25- T cells, in comparison with CD4+CD25+ regulatory T (Treg) cells was so far not thoroughly defined. Treg cells require a strong CD28 signal together with CD3Z for activation. We consequently used a CAR with combined CD28CD3Z signalling for redirecting CD4+CD25- T cells and CD4+CD25+ Treg cells from the same donor. CAR redirected activation of these T cell subsets and induced a distinct cytokine pattern with high IL-10 and a lack of IL-2 release by Treg cells. Despite strong antigen-specific activation, CAR Treg cells produced only weak target cell lysis, whereas CD4+CD25- CAR T cells were potent killers. Cytolysis did not correlate with the target cell sensitivity to Fas/FasL mediated killing; CD4+CD25- T cells upregulated perforin and granzyme B upon CAR activation, whereas Treg cells did less. The different cytolytic capacities of CAR redirected conventional CD4+ cells and Treg cells imply their use for different purposes in cell therapy. [ABSTRACT FROM AUTHOR]

Evidences are accumulating that CD4+ T cells can physiologically mediate antigen specific target cell lysis. By circumventing major histocompatibility complex (MHC)-restrictions through an engineered chimeric antigen receptor (CAR), CD4+ T cells lyse defined target cells as efficiently as do CD8+ T cells. However, the cytolytic capacity of redirected CD4+CD25− T cells, in comparison with CD4+CD25+ regulatory T (Treg) cells was so far not thoroughly defined. Treg cells require a strong CD28 signal together with CD3ζ for activation. We consequently used a CAR with combined CD28­CD3ζ signalling for redirecting CD4+CD25− T cells and CD4+CD25+ Treg cells from the same donor. CAR redirected activation of these T cell subsets and induced a distinct cytokine pattern with high IL-10 and a lack of IL-2 release by Treg cells. Despite strong antigen-specific activation, CAR Treg cells produced only weak target cell lysis, whereas CD4+CD25− CAR T cells were potent killers. Cytolysis did not correlate with the target cell sensitivity to Fas/FasL mediated killing; CD4+CD25− T cells upregulated perforin and granzyme B upon CAR activation, whereas Treg cells did less. The different cytolytic capacities of CAR redirected conventional CD4+ cells and Treg cells imply their use for different purposes in cell therapy.

Simple Summary: The ultimate goal of T cell-engaging immunotherapy is to endorse the activity of a person's own cytotoxic T cells in the tumor microenvironment, finally destroying cancer cells. Several types of immunotherapy are either approved for use or are under study in clinical trials to determine their effectiveness in treating various types of cancer. Chimeric antigen receptor (CAR) T cell therapy is rapidly emerging in the field and has shown unprecedented success in the treatment of hematological malignancies. It entails the collection of a patient's T cells to genetically engineer them in the lab to express CARs that target surface antigens on tumors to help identify and eradicate the tumor. However, major issues remain to be solved to enable generalized CART cell therapies in the clinic. Novel approaches to tackle these problems are being developed rapidly and are reviewed in this publication. In the past decade, chimeric antigen receptor (CAR) T cell technology has revolutionized cancer immunotherapy. This strategy uses synthetic CARs to redirect the patient's own immune cells to recognize specific antigens expressed on the surface of tumor cells. The unprecedented success of anti-CD19 CAR T cell therapy against B cell malignancies has resulted in its approval by the US Food and Drug Administration (FDA) in 2017. However, major scientific challenges still remain to be addressed for the broad use of CAR T cell therapy. These include severe toxicities, limited efficacy against solid tumors, and immune suppression in the hostile tumor microenvironment. Furthermore, CAR T cell therapy is a personalized medicine of which the production is time- and resource-intensive, which makes it very expensive. All these factors drive new innovations to engineer more powerful CAR T cells with improved antitumor activity, which are reviewed in this manuscript. [ABSTRACT FROM AUTHOR]

Simple Summary: In this study, we apply the ERBB2-chimeric antigen receptor (CAR)-modified natural killer (NK) cell line NK-92 (NK-92/5.28.z), a well-defined, good manufacturing practice (GMP)-compliant, third-party, off-the-shelf immune effector cell product as a novel immunotherapeutic approach for the treatment of high-risk rhabdomyosarcomas. Our preclinical in vitro data show enormous potential to improve immunotherapy of ERBB2-positive high-risk rhabdomyosarcoma a still incurable, rapidly lethal disease, assigning to NK-92/5.28.z cells rather than to unmodified parental NK-92 cells a multifarious role as ERBB2-specific CAR-targeted killers and modulators of endogenous adaptive immunity of the host, justifying the further evaluation of this approach in in vivo mouse xenograft models as a prerequisite for a possible future phase I/II clinical trial in defined subsets of high-risk rhabdomyosarcoma patients. The dismal prognosis of pediatric and young adult patients with high-risk rhabdomyosarcoma (RMS) underscores the need for novel treatment options for this patient group. In previous studies, the tumor-associated surface antigen ERBB2 (HER2/neu) was identified as targetable in high-risk RMS. As a proof of concept, in this study, a novel treatment approach against RMS tumors using a genetically modified natural killer (NK)-92 cell line (NK-92/5.28.z) as an off-the-shelf ERBB2-chimeric antigen receptor (CAR)-engineered cell product was preclinically explored. In cytotoxicity assays, NK-92/5.28.z cells specifically recognized and efficiently eliminated RMS cell suspensions, tumor cell monolayers, and 3D tumor spheroids via the ERBB2-CAR even at effector-to-target ratios as low as 1:1. In contrast to unmodified parental NK-92 cells, which failed to lyse RMS cells, NK-92/5.28.z cells proliferated and became further activated through contact with ERBB2-positive tumor cells. Furthermore, high amounts of effector molecules, such as proinflammatory and antitumoral cytokines, were found in cocultures of NK-92/5.28.z cells with tumor cells. Taken together, our data suggest the enormous potential of this approach for improving the immunotherapy of treatment-resistant tumors, revealing the dual role of NK-92/5.28.z cells as CAR-targeted killers and modulators of endogenous adaptive immunity even in the inhibitory tumor microenvironment of high-risk RMS. [ABSTRACT FROM AUTHOR]

Simple Summary: Interleukin-1 cytokines are key proinflammatory cytokines which have been implicated with differing pro- and antitumorigenic properties. Recent years have brought exciting insights and developments in IL-1-targeted therapies. Here, we present an overview of past and present research focusing on the role of IL-1 in cancer, with a special focus on clinical research and on therapeutic implications. With this, we strive to assist scientists in their future research objectives and to highlight possible directions for IL-1-targeting therapies in the coming years. Since its discovery, interleukin-1 has been extensively studied in a wide range of medical fields. Besides carrying out vital physiological functions, it has been implicated with a pivotal role in the progression and spreading of different cancer entities. During the last years, several clinical trials have been conducted, shedding light on the role of IL-1 blocking agents for the treatment of cancer. Additionally, recent developments in the field of immuno-oncology have implicated IL-1-induced signaling cascades as a major driver of severe chimeric antigen receptor T cell-associated toxicities such as cytokine release syndrome and immune effector cell-associated neurotoxicity. In this review, we summarize current clinical trials investigating the role of IL-1 blockade in cancer treatment and elaborate the proposed mechanism of these innovative treatment approaches. Additionally, we highlight cutting-edge developments utilizing IL-1 blocking agents to enhance the safety and efficacy of adoptive T cell therapy. [ABSTRACT FROM AUTHOR]

T cells engineered with chimeric antigen receptors (CARs) show great promise in the treatment of some cancers. Modifying T cells to express CARs generally relies on T-cell transduction using viral vectors carrying a transgene, resulting in semi-random DNA integration within the T-cell genome. While this approach has proven successful and is used in generating the Food and Drug Administration (FDA, USA) approved B-lymphocyte antigen CD19-specific CAR T cells, it is possible the transgene could integrate into a locus that would lead to malignant transformation of the engineered T cells. In addition, manufacturing viral vectors is time-consuming and expensive. One way to overcome these challenges is site-specific gene integration, which can be achieved through clustered regularly interspaced short palindromic repeat (CRISPR) mediated editing and non-viral DNA, which serves as a template for homology-directed repair (HDR). This non-viral gene editing approach provides a rapid, highly specific, and inexpensive way to engineer T cells. Here, we describe an optimized protocol for the site-specific knock-in of a large transgene in primary human T cells using non-viral double stranded DNA as a repair template. As proof-of-principle, we targeted the T-cell receptor alpha constant (TRAC) locus for insertion of a large transgene containing green fluorescence protein (GFP) and interleukin-15 (IL-15). To optimize the knock-in conditions we tested template DNA concentration, homology arm length, cell number, and knock-in efficiency over time. We then applied these established guidelines to target the TRAC or interleukin-13 (IL-13) locus for the knock-in of synthetic molecules, such as a CAR, bispecific T-cell engager (BiTE), and other transgenes. While integration efficiency depends on the targeted gene locus and selected transgene, this optimized protocol reliably generates the desired insertion at rates upwards of 20%. Thus, it should serve as a good starting point for investigators who are interested in knocking in transgenes into specific loci. [ABSTRACT FROM AUTHOR]

Oncolytic virotherapy is a novel approach to cancer therapy. Ad-fosARE is a conditionally replicative adenovirus engineered by inserting AU-rich elements (ARE) in the 3'-untranslated region of the E1A gene. In this study, we examined the oncolytic activity of Ad-fosARE and used it in a synergistic combination with the chemotherapeutic agent paclitaxel (PTX) for treating cancer cells. The expression of E1A was high in cancer cells due to stabilized E1A-ARE mRNA. As a result, the efficiency of its replication and cytolytic activity in cancer cells was higher than in normal cells. PTX treatment increased the cytoplasmic HuR relocalization in cancer cells, enhanced viral replication through elevated E1A expression, and upregulated CAR (Coxsackie-adenovirus receptor) required for viral uptake. Furthermore, PTX altered the instability of microtubules by acetylation and detyrosination, which is essential for viral internalization and trafficking to the nucleus. These results indicate that PTX can provide multiple advantages to the efficacy of Ad-fosARE both in vitro and in vivo, and provides a basis for designing novel clinical trials. Thus, this virus has a lot of benefits that are not found in other oncolytic viruses. The virus also has the potential for treating PXT-resistant cancers. [ABSTRACT FROM AUTHOR]

Evidences are accumulating that CD4⁺ T cells can physiologically mediate antigen specific target cell lysis. By circumventing major histocompatibility complex (MHC)-restrictions through an engineered chimeric antigen receptor (CAR), CD4⁺ T cells lyse defined target cells as efficiently as do CD8⁺ T cells. However, the cytolytic capacity of redirected CD4⁺CD25 - T cells, in comparison with CD4⁺CD25⁺ regulatory T (Treg) cells was so far not thoroughly defined. Treg cells require a strong CD28 signal together with CD3ζ for activation. We consequently used a CAR with combined CD28-CD3ζ signalling for redirecting CD4⁺CD25 - T cells and CD4⁺CD25⁺ Treg cells from the same donor. CAR redirected activation of these T cell subsets and induced a distinct cytokine pattern with high IL-10 and a lack of IL-2 release by Treg cells. Despite strong antigen-specific activation, CAR Treg cells produced only weak target cell lysis, whereas CD4⁺CD25 - CAR T cells were potent killers. Cytolysis did not correlate with the target cell sensitivity to Fas/FasL mediated killing; CD4⁺CD25 - T cells upregulated perforin and granzyme B upon CAR activation, whereas Treg cells did less. The different cytolytic capacities of CAR redirected conventional CD4⁺ cells and Treg cells imply their use for different purposes in cell therapy., Competing Interests: Authors declare not conflict of interest.