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NK cells are innate immune cells with inherent capabilities in both recognizing and killing cancer cells. NK cell phenotypes and functional alterations are being described with increasing precision among patients harboring various cancer types, emphasizing the critical role that NK cells play in antitumor immune responses. In addition, advances in understanding NK cell biology have improved our knowledge of such alterations, thereby expanding the potential exploitation of NK cells' anticancer capabilities. In this review, we present an overview of (1) the various types of NK cell alterations that may contribute to immune evasion in cancer patients and (2) the various strategies to improve NK cell-based anticancer immunotherapies, including pharmacologic modulation and/or genetic modification., (©2020 Society for Leukocyte Biology.)

T cells play a key role in tumour surveillance, both identifying and eliminating transformed cells. However, as tumours become established they form their own suppressive microenvironments capable of shutting down T cell function, and allowing tumours to persist and grow. To further understand the tumour microenvironment, including the interplay between different immune cells and their role in anti-tumour immune responses, a number of studies from mouse models to clinical trials have been performed. In this review, we examine mechanisms utilized by tumour cells to reduce their visibility to CD8 + Cytotoxic T lymphocytes (CTL), as well as therapeutic strategies trialled to overcome these tumour-evasion mechanisms. Next, we summarize recent advances in approaches to enhance CAR T cell activity and persistence over the past 10 years, including bispecific CAR T cell design and early evidence of efficacy. Lastly, we examine mechanisms of T cell infiltration and tumour regression, and discuss the strengths and weaknesses of different strategies to investigate T cell function in murine tumour models.

Approaches to harness the immune system to alleviate disease have become remarkably sophisticated since the crude, yet impressively-effective, attempts using live bacteria in the late 1800s. Recent evidence that engineered T cell therapy can deliver durable results in patients with cancer has spurred frenzied development in the field of T cell therapy. The myriad approaches include an innumerable variety of synthetic transgenes, multiplex gene-editing, and broader application to diseases beyond cancer. In this article, we review the preclinical studies and over a decade of clinical experience with engineered conventional T cells that have paved the way for translating engineered regulatory T cell therapies., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

Natural killer (NK) cells are lymphocytes with potent antitumor functions and, therefore, multiple NK cell-based cancer immunotherapies have been developed and are currently being tested. However, there is a necessity to find new means to improve these therapies, and immunometabolism represents an attractive target. NK cell effector functions are intricately linked to their metabolism, and modulating the latter could be the key to release their full potential. In this review, we have summarized how NK cell metabolism is regulated during some processes, such as maturation, viral infection, and cytokine stimulation. Additionally, we provide an overview of how NK cell metabolism is affected by current therapeutic approaches aimed to promote NK cell expansion and/or to increase their effector functions. We have also recapitulated several strategies that could help alleviating the metabolic impairment that characterizes tumor-infiltrating NK cells, and thus increase or restore their effector functions. Furthermore, we have reviewed several therapeutic approaches targeting cancer metabolism that could synergize with NK cell-based cancer immunotherapies, and thus enhance their efficacy., Competing Interests: Conflicts of interest The authors declare no competing interests., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

Natural killer (NK) cells are an important component of the innate immune system, particularly for metastasis immunosurveillance. They can rapidly recognize and kill transformed cells without the requirement of specific neo-antigen recognition. Their effector functions are modulated by a range of stimulatory and inhibitory surface receptors that regulate their cellular activation, differentiation and homeostasis. However, cancer cells can evade NK cell detection by receptor interaction or secretion of soluble immunosuppressant molecules. Therefore, genetic reprogramming of these immune suppressing or activating receptors of NK cells is a promising strategy to augment NK cell tumoricidal functions. In this review, we highlight the current clinical trials of chimeric antigen receptor engineered NK cells with redirected antigen specificity to eliminate hematological cancers and solid tumors. New alternative strategies that are advancing NK cell engineering for cancer treatment are also outlined. Lastly, different NK cell transgenesis approaches are reviewed and compared, and we discuss how these methods can be employed to maximize their anti-tumor effector functions., Competing Interests: Declaration of Competing Interest F.S.F.G. is a consultant for Biotheus Inc., (Crown Copyright © 2020. Published by Elsevier Inc. All rights reserved.)

Chimeric antigen receptor (CAR) T cell therapy has been successful in treating B cell malignancies in clinical trials; however, fewer studies have evaluated CAR T cell therapy for the treatment of T cell malignancies. There are many challenges in translating this therapy for T cell disease, including fratricide, T cell aplasia, and product contamination. To the best of our knowledge, no tumor-specific antigen has been identified with universal expression on cancerous T cells, hindering CAR T cell therapy for these malignancies. Numerous approaches have been assessed to address each of these challenges, such as (i) disrupting target antigen expression on CAR-modified T cells, (ii) targeting antigens with limited expression on T cells, and (iii) using third party donor cells that are either non-alloreactive or have been genome edited at the T cell receptor α constant (TRAC) locus. In this review, we discuss CAR approaches that have been explored both in preclinical and clinical studies targeting T cell antigens, as well as examine other potential strategies that can be used to successfully translate this therapy for T cell disease.

Cell-based therapies that employ engineered T cells-including those modified to express chimeric antigen receptors (CARs)-to target cancer cells have demonstrated promising responses in clinical trials. However, engineered T cell responses must be regulated to prevent severe side effects such as cytokine storms and off-target responses. Here we present a class of recombinase-based gene circuits that will enable inducible, one-time state switching in adoptive T cell therapy using an FDA-approved drug, creating a generalizable platform that can be used to control when and how strongly a gene is expressed. These circuits exhibit memory such that induced T cells will maintain any changes made even when the drug inducer is removed. This memory feature avoids prolonged drug inducer exposure, thus reducing the complexity and potential side effect associated with the drug inducer. We have utilized these circuits to control the expression of an anti-Her2-CAR, demonstrating the ability of these circuits to regulate CAR expression and T cell activity. We envision this platform can be extended to regulate other genes involved in T cell behavior for applications in various adoptive T cell therapies.

In recent years, the lymphocyte-specific protein tyrosine kinase (Lck) has emerged as one of the key molecules regulating T-cell functions. Studies using Lck knock-out mice or Lck-deficient T-cell lines have shown that Lck regulates the initiation of TCR signaling, T-cell development, and T-cell homeostasis. Because of the crucial role of Lck in T-cell responses, strategies have been employed to redirect Lck activity to improve the efficacy of chimeric antigen receptors (CARs) and to potentiate T-cell responses in cancer immunotherapy. In addition to the well-studied role of Lck in T cells, evidence has been accumulated suggesting that Lck is also expressed in the brain and in tumor cells, where it actively takes part in signaling processes regulating cellular functions like proliferation, survival and memory. Therefore, Lck has emerged as a novel druggable target molecule for the treatment of cancer and neuronal diseases. In this review, we will focus on these new functions of Lck., Competing Interests: The authors declare no conflict of interest.

Cytokines control immune-related events and are critically involved in a plethora of physiological and pathophysiological processes including autoimmunity and cancer development. Accordingly, modulation of natural cytokine signaling by antibodies and small molecules has improved therapeutic regimens. Synthetic biology sets out to optimize immunotherapeutics, with chimeric antigen receptor (CAR) T cell immmunotherapy being the first example to combine synthetic biology with genetic engineering during therapy. Hence, synthetic cytokines and cytokine receptors, as well as constitutively active cytokine receptor variants, are emerging as tools to improve or modulate immunotherapeutic strategies. This review focuses on recent developments in the growing field of synthetic cytokine signaling, providing an outlook for developing applications that involve physiological targets of immunotherapy., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Chimeric antigen receptor T-cell therapy has achieved highly promising results in clinical trials, particularly in B-cell malignancies. However, reports of serious adverse events including a number of patient deaths have raised concerns about safety of this treatment. Presently available preclinical models are not designed for predicting toxicities seen in human patients. Besides choosing the right animal model, careful considerations must be taken in chimeric antigen receptor T-cell design and the amount of T cells infused. The development of more sophisticated in vitro models and humanized mouse models for preclinical modeling and toxicity tests will help us to improve the design of clinical trials in cancer immunotherapy.

Glioblastoma multiforme (GBM) is the most malignant of the primary central nervous system (CNS) neoplasms, accounting for nearly 80% of all primary brain tumors and is associated with high morbidity and mortality. Immunotherapy is proving to be a fertile ground for next-generation GBM therapy, with large translational research projects and clinical trials currently underway. One particularly promising area is the chimeric antigen receptors (CARs) in the context of lymphocyte adoptive cell therapy (ACT), which has achieved success in the treatment of hematological malignancies. In this review, we will discuss CARs and review current challenges facing their use in GBM therapy. [Full article available at http://rimed.org/rimedicaljournal-2017-06.asp].

Effective clearance of virally infected cells requires the sequential activity of innate and adaptive immunity effectors. In human immunodeficiency virus (HIV) infection, naturally induced cell-mediated immune responses rarely eradicate infection. However, optimized immune responses could potentially be leveraged in HIV cure efforts if epitope escape and lack of sustained effector memory responses were to be addressed. Here we review leading HIV cure strategies that harness cell-mediated control against HIV in stably suppressed antiretroviral-treated subjects. We focus on strategies that may maximize target recognition and eradication by the sequential activation of a reconstituted immune system, together with delivery of optimal T-cell responses that can eliminate the reservoir and serve as means to maintain control of HIV spread in the absence of antiretroviral therapy (ART). As evidenced by the evolution of ART, we argue that a combination of immune-based strategies will be a superior path to cell-mediated HIV control and eradication. Available data from several human pilot trials already identify target strategies that may maximize antiviral pressure by joining innate and engineered T cell responses toward testing for sustained HIV remission and/or cure., (© The Author 2017. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.)

Patients with acute myeloid leukemia (AML) have relatively low survival rates compared to patients with other pediatric cancers. Relapse is frequent with conventional treatment and is a major cause of morbidity and mortality. Natural killer (NK) cells offer an alternative approach to chemotherapy that combats relapse by substantially eradicating AML blasts. New methods for enhancing NK cell activation and expression of the activating ligand on target malignant cells will increase the likelihood of success with this approach. We review these latest discoveries in NK cell-based therapy for AML and delineate recent advances in sensitizing AML cells to NK cell-mediated immunosurveillance., (© 2016 Wiley Periodicals, Inc.)

The consideration of the complex interplay between the tumour microenvironment (TME) and the immune response is the key for designing effective immunotherapies. Therapeutic strategies that harness co-stimulatory receptors have recently gained momentum in the clinic. One such strategy with promising clinical applications is the targeting of CD137, a member of the tumour necrosis factor receptor superfamily. Its expression on both innate and adaptive immune cells, coupled with its unique ability to potentiate antitumour responses through modulating the TME and to ameliorate autoimmune responses, has established it as an appealing target. In this review, we will discuss the various CD137-targeted immunotherapeutics that have reached clinical development, with a focus on recent advances and novel modalities such as CD137 chimeric antigen receptors and CD137 bispecific antibodies. We will also highlight the effect of CD137 targeting on the TME and discuss the importance of probing TME changes for predicting and testing the efficacy of CD137-mediated immunotherapy., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Tumours use many strategies to evade the host immune response, including downregulation or weak immunogenicity of target antigens and creation of an immune-suppressive tumour environment. T cells play a key role in cell-mediated immunity and, recently, strategies to genetically modify T cells either through altering the specificity of the T cell receptor (TCR) or through introducing antibody-like recognition in chimeric antigen receptors (CARs) have made substantial advances. The potential of these approaches has been demonstrated in particular by the successful use of genetically modified T cells to treat B cell haematological malignancies in clinical trials. This clinical success is reflected in the growing number of strategic partnerships in this area that have attracted a high level of investment and involve large pharmaceutical organisations. Although our understanding of the factors that influence the safety and efficacy of these therapies has increased, challenges for bringing genetically modified T-cell immunotherapy to many patients with different tumour types remain. These challenges range from the selection of antigen targets and dealing with regulatory and safety issues to successfully navigating the routes to commercial development. However, the encouraging clinical data, the progress in the scientific understanding of tumour immunology and the improvements in the manufacture of cell products are all advancing the clinical translation of these important cellular immunotherapies., (© 2015. Published by The Company of Biologists Ltd.)

Although the event-free survival for children and adolescents with acute lymphoblastic leukemia (ALL) has dramatically improved over the past half century, it has plateaued over the past decade. Children and adolescents with refractory/relapsed ALL continue to have a dismal prognosis with hematopoietic stem cell transplant being their most viable option for cure. There is an obvious need for the development of novel agents to further enhance overall outcomes. In this review we focus on the development of humoral and cellular immunotherapeutic agents in the treatment of childhood, adolescent, and young adult ALL. Immunotherapy in various forms has shown immense promise. To date we have seen numerous safety studies using monoclonal antibody therapy, antibody conjugates, bispecific T cell and bispecific natural killer (NK) cell antibodies and genetically reengineered T and NK cells expressing targeted chimeric antigen receptors. Initial success has been found with the anti-CD20 monoclonal antibodies followed by promising results using anti-CD22 and anti-CD19 therapies alone or in combination. Genetic modification of T and NK cells to express targeted chimeric antigen receptors offers a novel immunotherapy option that demonstrates enhanced cytotoxicity in otherwise resistant tumor cells. There is great potential to combine immunotherapies to further improve overall cure rates in children, adolescents, and young adults with poor-risk ALL. A number of humoral and cellular immunotherapy strategies have been investigated and found to be effective, safe, and well tolerated. Ideally, the targeted approach of immunotherapy will result in an overall decrease in toxicities experienced by patients. Future studies are required to determine when in the course of treatment with humoral and cellular therapy will have the safest and optimal effect in ALL., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Background Aims: Outcomes for patients with glioblastoma remain poor despite aggressive multimodal therapy. Immunotherapy with genetically modified T cells expressing chimeric antigen receptors (CARs) targeting interleukin (IL) 13Rα2, human epidermal growth factor receptor 2, epidermal growth factor variant III or erythropoietin-producing hepatocellular carcinoma A2 has shown promise for the treatment of glioma in preclinical models. On the basis of IL13Rα2 immunotoxins that contain IL13 molecules with one or two amino acid substitutions (IL13 muteins) to confer specificity to IL13Rα2, investigators have constructed CARS with IL13 muteins as antigen-binding domains. Whereas the specificity of IL13 muteins in the context of immunotoxins is well characterized, limited information is available for CAR T cells., Methods: We constructed four second-generation CARs with IL13 muteins with one or two amino acid substitutions, and evaluated the effector function of IL13-mutein CAR T cells in vitro and in vivo., Results: T cells expressing all four CARs recognized IL13Rα1 or IL13Rα2 recombinant protein in contrast to control protein (IL4R) as judged by interferon-γ production. IL13 protein produced significantly more IL2, indicating that IL13 mutein-CAR T cells have a higher affinity to IL13Rα2 than to IL13Rα1. In cytotoxicity assays, CAR T cells killed IL13Rα1- and/or IL13Rα2-positive cells in contrast to IL13Rα1- and IL13Rα2-negative controls. Although we observed no significant differences between IL13 mutein-CAR T cells in vitro, only T cells expressing IL13 mutein-CARs with an E13K amino acid substitution had anti-tumor activity in vivo that resulted in a survival advantage of treated animals., Conclusions: Our study highlights that the specificity/avidity of ligands is context-dependent and that evaluating CAR T cells in preclinical animal model is critical to assess their potential benefit., (Copyright © 2014 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.)

In the context of acute myeloid leukemia (AML) treatment, the interface between chemotherapy and immunotherapy is at present getting closer as never before. Scientific research is oriented in overcoming the main limits of actual chemotherapeutic regimens against AML, which still accounts for a considerable number of relapsed or resistant forms. A lot of investments have been done in the use of monoclonal antibodies (mAbs) and recently gene-modified immune cells have been considered as an alternative approach whenever chemotherapy fails to eradicate the disease. In this sense, AML is a potential suitable target for immunotherapeutic approaches, due to overexpression of several tumor antigens. Here we describe the state of the art of mAbs and cellular therapies employing engineered immune effectors, developed against specific AML antigens, in a window embracing preclinical research and translational studies to the clinical setting., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Chimeric antigen receptor (CAR) therapy has emerged as a ground-breaking advancement in cancer treatment, harnessing the power of engineered human immune cells to target and eliminate cancer cells. The escalating interest and investment in CAR therapy in recent years emphasize its profound significance in clinical research, positioning it as a rapidly expanding frontier in the field of personalized cancer therapies. A crucial step in CAR therapy design is choosing the right target as it determines the therapy's effectiveness, safety and specificity against cancer cells, while sparing healthy tissues. Herein, we propose a suite of tools for the identification and analysis of potential CAR targets leveraging expression data from The Cancer Genome Atlas and Genotype-Tissue Expression Project, which are implemented in CARTAR website. These tools focus on pinpointing tumor-associated antigens, ensuring target selectivity and assessing specificity to avoid off-tumor toxicities and can be used to rationally designing dual CARs. In addition, candidate target expression can be explored in cancer cell lines using the expression data for the Cancer Cell Line Encyclopedia. To our best knowledge, CARTAR is the first website dedicated to the systematic search of suitable candidate targets for CAR therapy. CARTAR is publicly accessible at https://gmxenomica.github.io/CARTAR/. [ABSTRACT FROM AUTHOR]

Chimeric antigen receptors (CARs) are genetically engineered receptors that recognize antigens and activate signaling cascades in a cell. Signal recognition and transmission are mediated by the CAR domains derived from different proteins. T cells carrying CARs against tumor-associated antigens have been used in the development of the CAR T cell therapy, a new approach to fighting malignant neoplasms. Despite its high efficacy in the treatment of oncohematological diseases, CAR T cell therapy has a number of disadvantages that could be avoided by using other types of leukocytes as effector cells. CARs can be expressed in a wide range of cells of adaptive and innate immunity with the emergence or improvement of cytotoxic properties. This review discusses the features of CAR function in different types of immune cells, with a particular focus on the results of preclinical and clinical efficacy studies and the safety of potential CAR cell products. [ABSTRACT FROM AUTHOR]

The human papillomavirus type 16 (HPV16) causes a large fraction of genital and oropharyngeal carcinomas. To maintain the transformed state, the tumor cells must continuously synthesize the E6 and E7 viral oncoproteins, which makes them tumor‐specific antigens. Indeed, specific T cell responses against them have been well documented and CD8+ T cells engineered to express T cell receptors (TCRs) that recognize epitopes of E6 or E7 have been tested in clinical studies with promising results, yet with limited clinical success. Using CD8+ T cells from peripheral blood of healthy donors, we have identified two novel TCRs reactive to an unexplored E618‐26 epitope. These TCRs showed limited standalone cytotoxicity against E618‐26‐HLA‐A*02:01‐presenting tumor cells. However, a single‐signaling domain chimeric antigen receptor (ssdCAR) targeting L1CAM, a cell adhesion protein frequently overexpressed in HPV16‐induced cancer, prompted a synergistic effect that significantly enhanced the cytotoxic capacity of NK‐92/CD3/CD8 cells armored with both TCR and ssdCAR when both receptors simultaneously engaged their respective targets, as shown by live microscopy of 2‐D and 3‐D co‐cultures. Thus, virus‐specific TCRs from the CD8+ T cell repertoire of healthy donors can be combined with a suitable ssdCAR to enhance the cytotoxic capacity of the effector cells and, indirectly, their specificity. [ABSTRACT FROM AUTHOR]

Background The epidemiology of cytomegalovirus (CMV) after chimeric antigen receptor–modified T-cell immunotherapy (CARTx) is poorly understood owing to a lack of routine surveillance. Methods We prospectively enrolled 72 adult CMV-seropositive CD19-, CD20-, or BCMA-targeted CARTx recipients and tested plasma samples for CMV before and weekly up to 12 weeks after CARTx. We assessed CMV-specific cell-mediated immunity (CMV-CMI) before and 2 and 4 weeks after CARTx, using an interferon γ release assay to quantify T-cell responses to IE-1 and pp65. We tested pre-CARTx samples to calculate a risk score for cytopenias and infection (CAR-HEMATOTOX). We used Cox regression to evaluate CMV risk factors and evaluated the predictive performance of CMV-CMI for CMV reactivation in receiver operator characteristic curves. Results CMV was detected in 1 patient (1.4%) before and in 18 (25%) after CARTx, for a cumulative incidence of 27% (95% confidence interval, 16.8–38.2). The median CMV viral load (interquartile range) was 127 (interquartile range, 61–276) IU/mL, with no end-organ disease observed; 5 patients received preemptive therapy based on clinical results. CMV-CMI values reached a nadir 2 weeks after infusion and recovered to baseline levels by week 4. In adjusted models, BCMA-CARTx (vs CD19/CD20) and corticosteroid use for >3 days were significantly associated with CMV reactivation, and possible associations were detected for lower week 2 CMV-CMI and more prior antitumor regimens. The cumulative incidence of CMV reactivation almost doubled when stratified by BCMA-CARTx target and use of corticosteroids for >3 days (46% and 49%, respectively). Conclusions CMV testing could be considered between 2 and 6 weeks in high-risk CARTx recipients. [ABSTRACT FROM AUTHOR]

Immunotherapy treatments aim to modulate the host's immune response to either mitigate it in inflammatory/autoimmune disease or enhance it against infection or cancer. Among different immunotherapies reaching clinical application during the last years, chimeric antigen receptor (CAR) immunotherapy has emerged as an effective treatment for cancer where different CAR T cells have already been approved. Yet their use against infectious diseases is an area still relatively poorly explored, albeit with tremendous potential for research and clinical application. Infectious diseases represent a global health challenge, with the escalating threat of antimicrobial resistance underscoring the need for alternative therapeutic approaches. This review aims to systematically evaluate the current applications of CAR immunotherapy in infectious diseases and discuss its potential for future applications. Notably, CAR cell therapies, initially developed for cancer treatment, are gaining recognition as potential remedies for infectious diseases. The review sheds light on significant progress in CAR T cell therapy directed at viral and opportunistic fungal infections. [ABSTRACT FROM AUTHOR]

We have developed a new format of a chimeric antigen receptor for αβ T cells, in which the single-chain variable fragment recognizing the tumor antigen is directly fused to the T cell receptor, called T cell receptor fusion construct (TRuC). Here, we express an anti-CD19 εTRuC in primary γδ T cells that were expanded using zoledronate (Zol) or concanavalin A. We show that the resulting εTRuC γδ T cells were reprogrammed to better recognize CD19-positive B cell tumors and—in case of the Zol-expanded cells—a CD19-expressing colon adenocarcinoma-derived cell line in vitro. This resulted in enhanced tumor killing, upregulation of the activation marker CD25, and secretion of cytokines. We found that the transduction efficiency of the concanavalin A–expanded cells was better than the one of the Zol-expanded ones. Our in vitro cytotoxicity data suggest that the Vδ2 T cells were better killers than the Vδ1 T cells. Finally, addition of vitamin C promoted the recovery of larger γδ T cell numbers after lentiviral transduction, as used for the expression of the εTRuC. In conclusion, the generation and use of γδ εTRuC T cells might be a new approach for cancer immunotherapy. [ABSTRACT FROM AUTHOR]

Abstract Background Even acknowledging the game-changing results achieved in the treatment of metastatic melanoma with the use of immune checkpoint inhibitors (ICI), a large proportion of patients (40–60%) still fail to respond or relapse due to the development of resistance. Alterations in the expression of Human Leukocyte Antigen class I (HLA-I) molecules are considered to play a major role in clinical resistance to ICI. Cellular immunotherapy with HLA-independent CAR-redirected lymphocytes is a promising alternative in this challenging setting and dedicated translational models are needed. Methods In this study, we propose an HLA-independent therapeutic strategy with Cytokine Induced Killer lymphocytes (CIK) genetically engineered with a Chimeric Antigen Receptor (CAR) targeting the tumor antigen CSPG4 as effector mechanism. We investigated the preclinical antitumor activity of CSPG4-CAR.CIK in vitro and in a xenograft murine model focusing on patient-derived melanoma cell lines (Mel) with defective expression of HLA-I molecules. Results We successfully generated CSPG4-CAR.CIK from patients with metastatic melanoma and reported their intense activity in vitro against a panel of CSPG4-expressing patient-derived Mel. The melanoma killing activity was intense, even at very low effector to target ratios, and not influenced by the expression level (high, low, defective) of HLA-I molecules on target cells. Furthermore, CAR.CIK conditioned medium was capable of upregulating the expression of HLA-I molecules on melanoma cells. A comparable immunomodulatory effect was replicated by treatment of Mel cells with exogenous IFN-γ and IFN-α. The antimelanoma activity of CSPG4-CAR.CIK was successfully confirmed in vivo, obtaining a significant tumor growth inhibition of an HLA-defective Mel xenograft in immunodeficient mice. Conclusions In this study we reported the intense preclinical activity of CSPG4-CAR.CIK against melanoma, including those with low or defective HLA-I expression. Our findings support CSPG4 as a valuable CAR target in melanoma and provide translational rationale for clinical studies exploring CAR-CIK cellular immunotherapies within the challenging setting of patients not responsive or relapsing to immune checkpoint inhibitors.

Background: Chimeric antigen receptor (CAR) T-cells have revolutionized the treatment of CD19- and B-cell maturation antigen-positive haematological malignancies. However, the effect of a CAR construct on the function of T-cells stimulated via their endogenous T-cell receptors (TCRs) has yet to be comprehensively investigated. Methods: Experiments were performed to systematically assess TCR signalling and function in CAR T-cells using anti-mesothelin human CAR T-cells as a model system. CAR T-cells expressing the CD28 or 4-1BB costimulatory endodomains were manufactured and compared to both untransduced T-cells and CAR T-cells with a non-functional endodomain. These cell products were treated with staphylococcal enterotoxin B to stimulate the TCR, and in vitro functional assays were performed by flow cytometry. Results: Increased proliferation, CD69 expression and IFNγ production were identified in CD8+ 4-1BBζ CAR T-cells compared to control untransduced CD8+ T-cells. These functional differences were associated with higher levels of phosphorylated ZAP70 after stimulation. In addition, these functional differences were associated with a differing immunophenotype, with a more than two-fold increase in central memory cells in CD8+ 4-1BBζ CAR T-cell products. Conclusion: Our data indicate that the 4-1BBζ CAR enhances CD8+ TCR-mediated function. This could be beneficial if the TCR targets epitopes on malignant tissues or infectious agents, but detrimental if the TCR targets autoantigens. [ABSTRACT FROM AUTHOR]

Background: Acute myeloid leukemia (AML) is a clonal malignant disorder which originates from a small number of leukemia-initiating cells or leukemic stem cells (LSCs)—the subpopulation that is also the root cause of relapsed/refractory AML. Chimeric antigen receptor (CAR)-T cell therapy has proved successful at combating certain hematologic malignancies, but has several hurdles that limit its widespread applications. CAR-natural killer (NK) cells do not carry the risk of inducing graft-versus-host disease (GvHD) frequently associated with allogeneic T cells, thereby overcoming time-consuming, autologous cell manufacturing, and have relatively safer clinical profiles than CAR-T cells. The present study aimed to generate anti-TIM3 CAR-NK cells targeting LSCs from a clonal master induced pluripotent stem cells engineered with the third-generation anti-TIM3 CAR. Methods: A clonal master umbilical cord blood NK-derived induced pluripotent stem cell (iPSC) line, MUSIi013-A, was used as a starting cells for engineering of an anti-TIM3 CAR harboring TIM3 scFv fragment (clone TSR-022), CD28, 4-1BB, and CD3ζ signaling (CAR-TIM3). The established CAR-TIM3 iPSCs were further differentiated under serum- and feeder-free conditions into functional CAR-TIM3 NK cells and tested for its anti-tumor activity against various TIM3-positive AML cells. Results: We successfully established a single-cell clone of CAR-TIM3 iPSCs, as validated by genomic DNA sequencing as well as antibody and antigen-specific detection. We performed thorough iPSC characterization to confirm its retained pluripotency and differentiation capacity. The established CAR-TIM3 iPSCs can be differentiated into CAR-TIM3 NK-like cells, which were further proven to have enhanced anti-tumor activity against TIM3-positive AML cells with minimal effect on TIM3-negative cells when compared with wild-type (WT) NK-like cells from parental iPSCs. Conclusions: iPSCs engineered with CARs, including the established single-cell clone CAR-TIM3 iPSCs herein, are potential alternative cell source for generating off-the-shelf CAR-NK cells as well as other CAR-immune cells. The feasibility of differentiation of functional CAR-TIM3 NK cells under serum- and feeder-free conditions support that Good Manufacturing Practice (GMP)-compliant protocols can be further established for future clinical applications. [ABSTRACT FROM AUTHOR]

Background: Even acknowledging the game-changing results achieved in the treatment of metastatic melanoma with the use of immune checkpoint inhibitors (ICI), a large proportion of patients (40–60%) still fail to respond or relapse due to the development of resistance. Alterations in the expression of Human Leukocyte Antigen class I (HLA-I) molecules are considered to play a major role in clinical resistance to ICI. Cellular immunotherapy with HLA-independent CAR-redirected lymphocytes is a promising alternative in this challenging setting and dedicated translational models are needed. Methods: In this study, we propose an HLA-independent therapeutic strategy with Cytokine Induced Killer lymphocytes (CIK) genetically engineered with a Chimeric Antigen Receptor (CAR) targeting the tumor antigen CSPG4 as effector mechanism. We investigated the preclinical antitumor activity of CSPG4-CAR.CIK in vitro and in a xenograft murine model focusing on patient-derived melanoma cell lines (Mel) with defective expression of HLA-I molecules. Results: We successfully generated CSPG4-CAR.CIK from patients with metastatic melanoma and reported their intense activity in vitro against a panel of CSPG4-expressing patient-derived Mel. The melanoma killing activity was intense, even at very low effector to target ratios, and not influenced by the expression level (high, low, defective) of HLA-I molecules on target cells. Furthermore, CAR.CIK conditioned medium was capable of upregulating the expression of HLA-I molecules on melanoma cells. A comparable immunomodulatory effect was replicated by treatment of Mel cells with exogenous IFN-γ and IFN-α. The antimelanoma activity of CSPG4-CAR.CIK was successfully confirmed in vivo, obtaining a significant tumor growth inhibition of an HLA-defective Mel xenograft in immunodeficient mice. Conclusions: In this study we reported the intense preclinical activity of CSPG4-CAR.CIK against melanoma, including those with low or defective HLA-I expression. Our findings support CSPG4 as a valuable CAR target in melanoma and provide translational rationale for clinical studies exploring CAR-CIK cellular immunotherapies within the challenging setting of patients not responsive or relapsing to immune checkpoint inhibitors. [ABSTRACT FROM AUTHOR]

Unlimited generation of chimeric antigen receptor (CAR) T cells from human-induced pluripotent stem cells (iPSCs) is an attractive approach for "off-the-shelf" CAR T cell immunotherapy. Approaches to efficiently differentiate iPSCs into canonical αβ T cell lineages, while maintaining CAR expression and functionality, however, have been challenging. We report that iPSCs reprogramed from CD62L+ naive and memory T cells followed by CD19-CAR engineering and 3D-organoid system differentiation confers products with conventional CD8αβ-positive CAR T cell characteristics. Expanded iPSC CD19-CAR T cells showed comparable antigen-specific activation, degranulation, cytotoxicity, and cytokine secretion compared with conventional CD19-CAR T cells and maintained homogeneous expression of the TCR derived from the initial clone. iPSC CD19-CAR T cells also mediated potent antitumor activity in vivo, prolonging survival of mice with CD19+ human tumor xenografts. Our study establishes feasible methodologies to generate highly functional CAR T cells from iPSCs to support the development of "off-the-shelf" manufacturing strategies.

Abstract Today, cancer treatment is one of the fundamental problems facing clinicians and researchers worldwide. Efforts to find an excellent way to treat this illness continue, and new therapeutic strategies are developed quickly. Adoptive cell therapy (ACT) is a practical approach that has been emerged to improve clinical outcomes in cancer patients. In the ACT, one of the best ways to arm the immune cells against tumors is by employing chimeric antigen receptors (CARs) via genetic engineering. CAR equips cells to target specific antigens on tumor cells and selectively eradicate them. Researchers have achieved promising preclinical and clinical outcomes with different cells by using CARs. One of the potent immune cells that seems to be a good candidate for CAR-immune cell therapy is the Natural Killer-T (NKT) cell. NKT cells have multiple features that make them potent cells against tumors and would be a powerful replacement for T cells and natural killer (NK) cells. NKT cells are cytotoxic immune cells with various capabilities and no notable side effects on normal cells. The current study aimed to comprehensively provide the latest advances in CAR-NKT cell therapy for cancers.

Immunotherapy treatments aim to modulate the host’s immune response to either mitigate it in inflammatory/autoimmune disease or enhance it against infection or cancer. Among different immunotherapies reaching clinical application during the last years, chimeric antigen receptor (CAR) immunotherapy has emerged as an effective treatment for cancer where different CAR T cells have already been approved. Yet their use against infectious diseases is an area still relatively poorly explored, albeit with tremendous potential for research and clinical application. Infectious diseases represent a global health challenge, with the escalating threat of antimicrobial resistance underscoring the need for alternative therapeutic approaches. This review aims to systematically evaluate the current applications of CAR immunotherapy in infectious diseases and discuss its potential for future applications. Notably, CAR cell therapies, initially developed for cancer treatment, are gaining recognition as potential remedies for infectious diseases. The review sheds light on significant progress in CAR T cell therapy directed at viral and opportunistic fungal infections.

Part of the broader glycosphingolipid family, gangliosides are composed of a ceramide bound to a sialic acid-containing glycan chain, and locate at the plasma membrane. Gangliosides are produced through sequential steps of glycosylation and sialylation. This diversity of composition is reflected in differences in expression patterns and functions of the various gangliosides. Ganglioside GD2 designates different subspecies following a basic structure containing three carbohydrate residues and two sialic acids. GD2 expression, usually restrained to limited tissues, is frequently altered in various neuroectoderm-derived cancers. While GD2 is of evident interest, its glycolipid nature has rendered research challenging. Physiological GD2 expression has been linked to developmental processes. Passing this stage, varying levels of GD2, physiologically expressed mainly in the central nervous system, affect composition and formation of membrane microdomains involved in surface receptor signaling. Overexpressed in cancer, GD2 has been shown to enhance cell survival and invasion. Furthermore, binding of antibodies leads to immune-independent cell death mechanisms. In addition, GD2 contributes to T-cell dysfunction, and functions as an immune checkpoint. Given the cancer-associated functions, GD2 has been a source of interest for immunotherapy. As a potential biomarker, methods are being developed to quantify GD2 from patients' samples. In addition, various therapeutic strategies are tested. Based on initial success with antibodies, derivates such as bispecific antibodies and immunocytokines have been developed, engaging patient immune system. Cytotoxic effectors or payloads may be redirected based on anti-GD2 antibodies. Finally, vaccines can be used to mount an immune response in patients. We review here the pertinent biological information on GD2 which may be of use for optimizing current immunotherapeutic strategies. [ABSTRACT FROM AUTHOR]

Advanced gene transfer technologies and profound immunological insights have enabled substantial increases in the efficacy of anticancer adoptive cellular therapy (ACT). In recent years, the U.S. Food and Drug Administration and European Medicines Agency have approved six engineered T cell therapeutic products, all chimeric antigen receptor-engineered T cells directed against B cell malignancies. Despite encouraging clinical results, engineered T cell therapy is still constrained by challenges, which could be addressed by genome editing. As RNA-guided Clustered Regularly Interspaced Short Palindromic Repeats technology passes its 10-year anniversary, we review emerging applications of genome editing approaches designed to (1) overcome resistance to therapy, including cancer immune evasion mechanisms; (2) avoid unwanted immune reactions related to allogeneic T cell products; (3) increase fitness, expansion capacity, persistence, and potency of engineered T cells, while preserving their safety profile; and (4) improve the ability of therapeutic cells to resist immunosuppressive signals active in the tumor microenvironment. Overall, these innovative approaches should widen the safe and effective use of ACT to larger number of patients affected by cancer. [ABSTRACT FROM AUTHOR]

Chimeric antigen receptors (CARs) are widely used by T cells (CAR-T cells), natural killer cells dendritic cells and macrophages, and they are of great importance in cellular immunotherapy. However, the use of CAR-related products faces several challenges, including the poor persistence of cells carrying CARs, cell dysfunction or exhaustion, relapse of disease, immune effector cell-associated neurotoxicity syndrome, cytokine release syndrome, low efficacy against solid tumors and immunosuppression by the tumor microenvironment. Another important cell therapy regimen involves mesenchymal stem cells (MSCs). Recent studies have shown that MSCs can improve the anticancer functions of CAR-related products. CAR-MSCs can overcome the flaws of cellular immunotherapy. Thus, MSCs can be used as a biological vehicle for CARs. In this review, we first discuss the characteristics and immunomodulatory functions of MSCs. Then, the role of MSCs as a source of exosomes, including the characteristics of MSC-derived exosomes and their immunomodulatory functions, is discussed. The role of MSCs in CAR-related products, CAR-related product-derived exosomes and the effect of MSCs on CAR-related products are reviewed. Finally, the use of MSCs as CAR vehicles is discussed. 4iUUHD81rmzA1EBQmMyRQx Video Abstract [ABSTRACT FROM AUTHOR]

Abstract Cancer immunotherapies utilizing genetically engineered T cells have emerged as powerful personalized therapeutic agents showing dramatic preclinical and clinical results, particularly in hematological malignancies. Ectopically expressed chimeric antigen receptors (CARs) reprogram immune cells to target and eliminate cancer. However, CAR T cell therapy's success depends on the balance between effective anti-tumor activity and minimizing harmful side effects. To improve CAR T cell therapy outcomes and mitigate associated toxicities, scientists from different fields are cooperating in developing next-generation products using the latest molecular cell biology and synthetic biology tools and technologies. The immunotherapy field is rapidly evolving, with new approaches and strategies being reported at a fast pace. This comprehensive literature review aims to provide an up-to-date overview of the latest developments in controlling CAR T cell activity for improved safety, efficacy, and flexibility.

Abstract Locoregional delivery of chimeric antigen receptor (CAR)-modified T (CAR-T) cells has emerged as a promising strategy for brain tumors. However, the complicated ex vivo cell manufacturing procedures and the rapid progression of the disease have limited its broader applications. Macrophages (MΦs) exhibit unique effector functions and a high degree of infiltration within the solid tumor microenvironment (TME), especially in the brain, where MΦs function as structural support, and the main immune effector cells of the CNS represent 5–12% of brain cells. Here, we report a synthetic universal DNA nanocarrier for in situ genetic editing of intratumoral MΦs with an ErbB2-specific CAR to direct their phagocytic activity towards tumors and subsequently initiate a locoregional antitumor immune response. Specifically, we demonstrated that when delivered locoregionally, the RP-182 peptide, located in the shell of a nanoparticle, targeted MΦs and reprogrammed M2-like tumor-associated macrophages (TAMs) to an antitumor M1-like phenotype. Subsequently, the CAR gene-laden DNA nanocomplex can be used to introduce ErbB2-targeted CAR, and the generated CAR-MΦs then act as “living” cures, thereby serially clearing the invasive tumor cells. Our work demonstrates a practical antitumor immunotherapy for brainstem gliomas (BSGs) that may be broadly applicable for patients suffering from other ErbB2-positive solid malignancies.

Part of the broader glycosphingolipid family, gangliosides are composed of a ceramide bound to a sialic acid-containing glycan chain, and locate at the plasma membrane. Gangliosides are produced through sequential steps of glycosylation and sialylation. This diversity of composition is reflected in differences in expression patterns and functions of the various gangliosides. Ganglioside GD2 designates different subspecies following a basic structure containing three carbohydrate residues and two sialic acids. GD2 expression, usually restrained to limited tissues, is frequently altered in various neuroectoderm-derived cancers. While GD2 is of evident interest, its glycolipid nature has rendered research challenging. Physiological GD2 expression has been linked to developmental processes. Passing this stage, varying levels of GD2, physiologically expressed mainly in the central nervous system, affect composition and formation of membrane microdomains involved in surface receptor signaling. Overexpressed in cancer, GD2 has been shown to enhance cell survival and invasion. Furthermore, binding of antibodies leads to immune-independent cell death mechanisms. In addition, GD2 contributes to T-cell dysfunction, and functions as an immune checkpoint. Given the cancer-associated functions, GD2 has been a source of interest for immunotherapy. As a potential biomarker, methods are being developed to quantify GD2 from patients’ samples. In addition, various therapeutic strategies are tested. Based on initial success with antibodies, derivates such as bispecific antibodies and immunocytokines have been developed, engaging patient immune system. Cytotoxic effectors or payloads may be redirected based on anti-GD2 antibodies. Finally, vaccines can be used to mount an immune response in patients. We review here the pertinent biological information on GD2 which may be of use for optimizing current immunotherapeutic strategies.

There is a medical need to develop new and effective therapies against triple-negative breast cancer (TNBC). Chimeric antigen receptor (CAR) natural killer (NK) cells are a promising alternative to CAR-T cell therapy for cancer. A search for a suitable target in TNBC identified CD44v6, an adhesion molecule expressed in lymphomas, leukemias and solid tumors that is implicated in tumorigenesis and metastases. We have developed a next-generation CAR targeting CD44v6 that incorporates IL-15 superagonist and checkpoint inhibitor molecules. We could show that CD44v6 CAR-NK cells demonstrated effective cytotoxicity against TNBC in 3D spheroid models. The IL-15 superagonist was specifically released upon recognition of CD44v6 on TNBC and contributed to the cytotoxic attack. PD1 ligands are upregulated in TNBC and contribute to the immunosuppressive tumor microenvironment (TME). Competitive inhibition of PD1 neutralized inhibition by PD1 ligands expressed on TNBC. In total, CD44v6 CAR-NK cells are resistant to TME immunosuppression and offer a new therapeutic option for the treatment of BC, including TNBC. [ABSTRACT FROM AUTHOR]

Today, cancer treatment is one of the fundamental problems facing clinicians and researchers worldwide. Efforts to find an excellent way to treat this illness continue, and new therapeutic strategies are developed quickly. Adoptive cell therapy (ACT) is a practical approach that has been emerged to improve clinical outcomes in cancer patients. In the ACT, one of the best ways to arm the immune cells against tumors is by employing chimeric antigen receptors (CARs) via genetic engineering. CAR equips cells to target specific antigens on tumor cells and selectively eradicate them. Researchers have achieved promising preclinical and clinical outcomes with different cells by using CARs. One of the potent immune cells that seems to be a good candidate for CAR-immune cell therapy is the Natural Killer-T (NKT) cell. NKT cells have multiple features that make them potent cells against tumors and would be a powerful replacement for T cells and natural killer (NK) cells. NKT cells are cytotoxic immune cells with various capabilities and no notable side effects on normal cells. The current study aimed to comprehensively provide the latest advances in CAR-NKT cell therapy for cancers. [ABSTRACT FROM AUTHOR]

Glioblastoma remains as the most common and aggressive primary adult brain tumor to date. Within the last decade, cancer immunotherapy surfaced as a broadly successful therapeutic approach for a variety of cancers. However, due to the neuroanatomical and immunosuppressive nature of malignant gliomas, conventional chemotherapy and radiotherapy treatments garner limited efficacy in patients with these tumors. The intricate structure of the blood brain barrier restricts immune accessibility into the tumor microenvironment, and malignant gliomas can activate various adaptive responses to subvert anticancer immune responses and reinstate an immunosuppressive milieu. Yet, evidence of lymphocyte infiltration within the brain and recent advancements made in cell engineering technologies implicate the vast potential in the future of neuro-oncological immunotherapy. Previous immunotherapy platforms have paved way to improved modalities, which includes but is not limited to personalized vaccines and chimeric antigen receptor T-cell therapy. This review will cover the various neuroanatomical and immunosuppressive features of central nervous system tumors and highlight the innovations made in T-cell based therapies to overcome the challenges presented by the glioblastoma microenvironment.

Abstract Background Aberrant activation of the MET receptor in cancer is sustained by genetic alterations or, more frequently, by transcriptional upregulations. A fraction of MET-amplified or mutated tumors are sensible to MET targeting agents, but their responsiveness is typically short-lasting, as secondary resistance eventually occurs. Since in the absence of genetic alterations MET is usually not a tumor driver, MET overexpressing tumors are not/poorly responsive to MET targeted therapies. Consequently, the vast majority of tumors exhibiting MET activation still represent an unmet medical need. Methods Here we propose an immunotherapy strategy based on T lymphocytes expressing a Chimeric Antigen Receptor (CAR) targeting MET overexpressing tumors of different histotypes. We engineered two different MET-CAR constructs and tested MET-CAR-T cell cytotoxic activity against different MET overexpressing models, including tumor cell lines, primary cancer cells, organoids, and xenografts in immune-deficient mice. Results We proved that MET-CAR-T exerted a specific cytotoxic activity against MET expressing cells. Cell killing was proportional to the level of MET expressed on the cell surface. While CAR-T cytotoxicity was minimal versus cells carrying MET at physiological levels, essentially sparing normal cells, the activity versus MET overexpressing tumors was robust, significantly controlling tumor cell growth in vitro and in vivo. Notably, MET-CAR-T cells were also able to brake acquired resistance to MET targeting agents in MET amplified cancer cells carrying secondary mutations in downstream signal transducers. Conclusions We set and validated at the pre-clinical level a MET-CAR immunotherapy strategy potentially beneficial for cancers not eligible for MET targeted therapy with inhibitory molecules, including those exhibiting primary or secondary resistance.

Chimeric antigen receptor (CAR) T cell therapy has demonstrated unprecedented success with high remission rates for heavily pretreated patients with hematological malignancies. The hinge connecting the extracellular antigen recognition unit to the transmembrane domain provides the length and flexibility of the CAR constructs and ensures that the CAR can reach the target antigen and mediate recognition and killing of target cells. The hinge can also include specific amino acid sequences to improve CAR expression, influence T cell proliferation, and facilitate CAR T cell detection, enrichment, and even elimination. Here, we report the generation of two novel hinge domains derived from the low-affinity p75 chain of the human nerve growth factor receptor (NGFR), termed N3 and N4, which, when incorporated into the CAR backbone, allow detection as well as high-grade enrichment of CAR T cells with GMP-compatible immunomagnetic reagents. After optimizing the MACS protocol for excellent CAR T cell purity and yield, we demonstrated that N3- and N4-hinged CAR T cells are as efficacious as their CD8-hinged counterparts in vitro against hematological blasts and also in vivo in the control of acute monocytic leukemia in an immunodeficient mouse xenograft model. Thus, both hinges could potentially be an integral part of future CAR designs and universally applicable in clinical applications.

Cancer immunotherapies utilizing genetically engineered T cells have emerged as powerful personalized therapeutic agents showing dramatic preclinical and clinical results, particularly in hematological malignancies. Ectopically expressed chimeric antigen receptors (CARs) reprogram immune cells to target and eliminate cancer. However, CAR T cell therapy's success depends on the balance between effective anti-tumor activity and minimizing harmful side effects. To improve CAR T cell therapy outcomes and mitigate associated toxicities, scientists from different fields are cooperating in developing next-generation products using the latest molecular cell biology and synthetic biology tools and technologies. The immunotherapy field is rapidly evolving, with new approaches and strategies being reported at a fast pace. This comprehensive literature review aims to provide an up-to-date overview of the latest developments in controlling CAR T cell activity for improved safety, efficacy, and flexibility. [ABSTRACT FROM AUTHOR]

Ovarian cancer (OC) is among the most common gynecologic malignancies with a poor prognosis and a high mortality rate. Most patients are diagnosed at an advanced stage (stage III or IV), with 5-year survival rates ranging from 25% to 47% worldwide. Surgical resection and first-line chemotherapy are the main treatment modalities for OC. However, patients usually relapse within a few years of initial treatment due to resistance to chemotherapy. Cell-based therapies, particularly adoptive T-cell therapy and chimeric antigen receptor T (CAR-T) cell therapy, represent an alternative immunotherapy approach with great potential for hematologic malignancies. However, the use of CAR-T-cell therapy for the treatment of OC is still associated with several difficulties. In this review, we comprehensively discuss recent innovations in CAR-T-cell engineering to improve clinical efficacy, as well as strategies to overcome the limitations of CAR-T-cell therapy in OC. [ABSTRACT FROM AUTHOR]

Locoregional delivery of chimeric antigen receptor (CAR)-modified T (CAR-T) cells has emerged as a promising strategy for brain tumors. However, the complicated ex vivo cell manufacturing procedures and the rapid progression of the disease have limited its broader applications. Macrophages (MΦs) exhibit unique effector functions and a high degree of infiltration within the solid tumor microenvironment (TME), especially in the brain, where MΦs function as structural support, and the main immune effector cells of the CNS represent 5–12% of brain cells. Here, we report a synthetic universal DNA nanocarrier for in situ genetic editing of intratumoral MΦs with an ErbB2-specific CAR to direct their phagocytic activity towards tumors and subsequently initiate a locoregional antitumor immune response. Specifically, we demonstrated that when delivered locoregionally, the RP-182 peptide, located in the shell of a nanoparticle, targeted MΦs and reprogrammed M2-like tumor-associated macrophages (TAMs) to an antitumor M1-like phenotype. Subsequently, the CAR gene-laden DNA nanocomplex can be used to introduce ErbB2-targeted CAR, and the generated CAR-MΦs then act as "living" cures, thereby serially clearing the invasive tumor cells. Our work demonstrates a practical antitumor immunotherapy for brainstem gliomas (BSGs) that may be broadly applicable for patients suffering from other ErbB2-positive solid malignancies. [ABSTRACT FROM AUTHOR]