Search

Natural Killer (NK) cells are exciting candidates for cancer immunotherapy with potent innate cytotoxicity and distinct advantages over T cells for Chimeric Antigen Receptor (CAR) therapy. Concerns regarding the safety, cost, and scalability of viral vectors has ignited research into non-viral alternatives for gene delivery. This review comprehensively analyses recent advancements and challenges with non-viral genetic modification of NK cells for allogeneic CAR-NK therapies. Non-viral alternatives including electroporation and multifunctional nanoparticles are interrogated with respect to CAR expression and translational responses. Crucially, the link between NK cell biology and design of drug delivery technologies are made, which is essential for development of future non-viral approaches. This review provides valuable insights into the current state of non-viral CAR-NK cell engineering, aimed at realising the full potential of NK cell-based immunotherapies., (© 2024. The Author(s).)

CD19-targeted chimeric antigen receptor (CAR) T cell therapies have driven a paradigm shift in the treatment of relapsed/refractory B-cell malignancies. However, >50% of CD19-CAR-T-treated patients experience progressive disease mainly due to antigen escape and low persistence. Clinical prognosis is heavily influenced by CAR-T cell function and systemic cytokine toxicities. Furthermore, it remains a challenge to efficiently, cost-effectively, and consistently manufacture clinically relevant numbers of virally engineered CAR-T cells. Using a highly efficient piggyBac transposon-based vector, Quantum pBac™ (qPB), we developed a virus-free cell-engineering system for development and production of multiplex CAR-T therapies. Here, we demonstrate in vitro and in vivo that consistent, robust and functional CD20/CD19 dual-targeted CAR-T stem cell memory (CAR-TSCM) cells can be efficiently produced for clinical application using qPB™. In particular, we showed that qPB™-manufactured CAR-T cells from cancer patients expanded efficiently, rapidly eradicated tumors, and can be safely controlled via an iCasp9 suicide gene-inducing drug. Therefore, the simplicity of manufacturing multiplex CAR-T cells using the qPB™ system has the potential to improve efficacy and broaden the accessibility of CAR-T therapies., Competing Interests: GenomeFrontier Therapeutics TW Co., Ltd. funded the study and played a crucial role in the study design, data collection and analysis, decision to publish, and preparation of the manuscripts. The coauthors – Peter S. Chang, Yi-Chun Chen, Wei-Kai Hua, Jeff C. Hsu, Jui-Cheng Tsai, Yi-Wun Huang, Yi-Hsin Kao, Pei-Hua Wu, Kuo-Lan Karen Wen, and Sareina Chiung-Yuan Wu – are affiliated with GenomeFrontier Therapeutics TW Co., Ltd. This does not alter our adherence to PLOS ONE policies on sharing data and materials., (Copyright: © 2024 Chang et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Natural killer (NK) cells have high intrinsic cytotoxic capacity, and clinical trials have demonstrated their safety and efficacy for adoptive cancer therapy. Expression of chimeric antigen receptors (CARs) enhances NK cell target specificity, with these cells applicable as off-the-shelf products generated from allogeneic donors. Here, we present for the first time an innovative approach for CAR NK cell engineering employing a non-viral Sleeping Beauty (SB) transposon/transposase-based system and minimized DNA vectors termed minicircles. SB-modified peripheral blood-derived primary NK cells displayed high and stable CAR expression and more frequent vector integration into genomic safe harbors than lentiviral vectors. Importantly, SB-generated CAR NK cells demonstrated enhanced cytotoxicity compared with non-transfected NK cells. A strong antileukemic potential was confirmed using established acute lymphocytic leukemia cells and patient-derived primary acute B cell leukemia and lymphoma samples as targets in vitro and in vivo in a xenograft leukemia mouse model. Our data suggest that the SB-transposon system is an efficient, safe, and cost-effective approach to non-viral engineering of highly functional CAR NK cells, which may be suitable for cancer immunotherapy of leukemia as well as many other malignancies., Competing Interests: Declaration of interests E.U. is an Advisory Board member for Phialogics and has sponsored research projects with Gilead and BMS. Z.I. is an inventor on patents related to Sleeping Beauty and MC technology. M.H. is listed as inventor on patent applications and granted patents related to CAR technologies and transposon-based gene transfer that are, in part licensed to industry. M.H. is a co-founder and equity owner of T-CURX GmbH, Würzburg, Germany. M.H. and E.U. are inventors on patents related to CAR and MC technology. T.B., P.W., W.S.W., and E.U. are inventors on patents related to optimized CAR designs. T.B., P.W., and E.U. are inventors on non-viral gene-editing technologies of NK cells., (Copyright © 2024. Published by Elsevier Inc.)

T cells modified to express intelligently designed chimeric antigen receptors (CARs) are exceptionally powerful therapeutic agents for relapsed and refractory blood cancers and have the potential to revolutionize therapy for many other diseases. To circumvent the complexity and cost associated with broad-scale implementation of ex vivo manufactured adoptive cell therapy products, alternative strategies to generate CAR T cells in vivo by direct infusion of nanoparticle-formulated nucleic acids or engineered viral vectors under development have received a great deal of attention in the past few years. Here, we outline the ex vivo manufacturing process as a motivating framework for direct in vivo strategies and discuss emerging data from preclinical models to highlight the potency of the in vivo approach, the applicability for new disease indications, and the remaining challenges associated with clinical readiness, including delivery specificity, long term efficacy, and safety., Competing Interests: Declaration of interests PRC has financial interests in Acuitas Therapeutics, Mesentech, and NanoVation Therapeutics. LE collaborates with NanoVation Therapeutics and has received material support. RAH and LS do not have any conflicts to disclose., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Although chimeric antigen receptor (CAR) T cells have altered the treatment landscape for B cell malignancies, the risk of on-target, off-tumour toxicity has hampered their development for solid tumours because most target antigens are shared with normal cells 1,2 . Researchers have attempted to apply Boolean-logic gating to CAR T cells to prevent toxicity 3-5 ; however, a truly safe and effective logic-gated CAR has remained elusive 6 . Here we describe an approach to CAR engineering in which we replace traditional CD3ζ domains with intracellular proximal T cell signalling molecules. We show that certain proximal signalling CARs, such as a ZAP-70 CAR, can activate T cells and eradicate tumours in vivo while bypassing upstream signalling proteins, including CD3ζ. The primary role of ZAP-70 is to phosphorylate LAT and SLP-76, which form a scaffold for signal propagation. We exploited the cooperative role of LAT and SLP-76 to engineer logic-gated intracellular network (LINK) CAR, a rapid and reversible Boolean-logic AND-gated CAR T cell platform that outperforms other systems in both efficacy and prevention of on-target, off-tumour toxicity. LINK CAR will expand the range of molecules that can be targeted with CAR T cells, and will enable these powerful therapeutic agents to be used for solid tumours and diverse diseases such as autoimmunity 7 and fibrosis 8 . In addition, this work shows that the internal signalling machinery of cells can be repurposed into surface receptors, which could open new avenues for cellular engineering., (© 2023. The Author(s), under exclusive licence to Springer Nature Limited.)

Natural killer cells (NK cells) are the first line of the innate immune defense system, primarily located in peripheral circulation and lymphoid tissues. They kill virally infected and malignant cells through a balancing play of inhibitory and stimulatory receptors. In pre-clinical investigational studies, NK cells show promising anti-tumor effects and are used in adoptive transfer of activated and expanded cells, ex-vivo . NK cells express co-stimulatory molecules that are attractive targets for the immunotherapy of cancers. Recent clinical trials are investigating the use of CAR-NK for different cancers to determine the efficiency. Herein, we review NK cell therapy approaches (NK cell preparation from tissue sources, ways of expansion ex-vivo for "off-the-shelf" allogeneic cell-doses for therapies, and how different vector delivery systems are used to engineer NK cells with CARs) for cancer immunotherapy., Competing Interests: EH, EZ, TS-N, AH, SB, NK, and MV had compensated employment at Thermo Fisher Scientific., (Copyright © 2021 Heipertz, Zynda, Stav-Noraas, Hungler, Boucher, Kaur and Vemuri.)

Spearheaded by the therapeutic use of chimeric antigen receptors (CARs) targeting CD19, synthetic immunology has entered the clinical arena. CARs are recombinant receptors for antigen that engage cell surface molecules through the variable region of an antibody and signal through arrayed T-cell activating and costimulatory domains. CARs allow redirection of T-cell cytotoxicity against any antigen of choice, independent of MHC expression. Patient T cells engineered to express CARs specific for CD19 have yielded remarkable outcomes in subjects with relapsed/refractory B- cell malignancies, setting off unprecedented interest in T-cell engineering and cell-based cancer immunotherapy. In this review, we present the challenges to extend the use of CAR T cells to solid tumors and other pathologies. We further highlight progress in CAR design, cell manufacturing, and genome editing, which in aggregate hold the promise of generating safer and more effective genetically instructed immunity. Novel engineered cell types, including innate T-cell types, natural killer (NK) cells, macrophages, and induced pluripotent stem cell-derived immune cells, are on the horizon, as are applications of CAR T cells to treat autoimmunity, severe infections, and senescence-associated pathologies., (© 2021 Wiley-VCH GmbH.)

Targeting T cell malignancies with universal CD7-targeting chimeric antigen receptor T cells (UCART7) can lead to profound immune deficiency due to loss of normal T and NK cells. While a small population of endogenous CD7- T cells exists, these cells are unlikely to be able to repopulate the entire immune repertoire after UCART7 treatment, as they are limited in number and proliferative capacity. To rescue T and NK cells after UCART7, we created hematopoietic stem cells genetically deleted for CD7 (CD7-KO HSCs). CD7-KO HSCs were able to engraft immunodeficient mice and differentiate into T and NK cells lacking CD7 expression. CD7-KO T and NK cells could perform effector functions as robustly as control T and NK cells. Furthermore, CD7-KO T cells were phenotypically and functionally distinct from endogenous CD7- T cells, indicating that CD7-KO T cells can supplement immune functions lacking in CD7- T cells. Mice engrafted with CD7-KO HSCs maintained T and NK cell numbers after UCART7 treatment, while these were significantly decreased in control mice. These studies support the development of CD7-KO HSCs to augment host immunity in patients with T cell malignancies after UCART7 treatment.

Adenosine is an immunosuppressive factor that limits anti-tumor immunity through the suppression of multiple immune subsets including T cells via activation of the adenosine A 2A receptor (A 2A R). Using both murine and human chimeric antigen receptor (CAR) T cells, here we show that targeting A 2A R with a clinically relevant CRISPR/Cas9 strategy significantly enhances their in vivo efficacy, leading to improved survival of mice. Effects evoked by CRISPR/Cas9 mediated gene deletion of A 2A R are superior to shRNA mediated knockdown or pharmacological blockade of A 2A R. Mechanistically, human A 2A R-edited CAR T cells are significantly resistant to adenosine-mediated transcriptional changes, resulting in enhanced production of cytokines including IFNγ and TNF, and increased expression of JAK-STAT signaling pathway associated genes. A 2A R deficient CAR T cells are well tolerated and do not induce overt pathologies in mice, supporting the use of CRISPR/Cas9 to target A 2A R for the improvement of CAR T cell function in the clinic.

Checkpoint inhibitors and T-cell therapies have highlighted the critical role of T cells in anti-cancer immunity. However, limitations associated with these treatments drive the need for alternative approaches. Here, we engineer red blood cells into artificial antigen-presenting cells (aAPCs) presenting a peptide bound to the major histocompatibility complex I, the costimulatory ligand 4-1BBL, and interleukin (IL)-12. This leads to robust, antigen-specific T-cell expansion, memory formation, additional immune activation, tumor control, and antigen spreading in tumor models in vivo. The presence of 4-1BBL and IL-12 induces minimal toxicities due to restriction to the vasculature and spleen. The allogeneic aAPC, RTX-321, comprised of human leukocyte antigen-A*02:01 presenting the human papilloma virus (HPV) peptide HPV16 E7 11-19 , 4-1BBL, and IL-12 on the surface, activates HPV-specific T cells and promotes effector function in vitro. Thus, RTX-321 is a potential 'off-the-shelf' in vivo cellular immunotherapy for treating HPV + cancers, including cervical and head/neck cancers.

The immune system is a sophisticated network of different cell types performing complex biocomputation at single-cell and consortium levels. The ability to reprogram such an interconnected multicellular system holds enormous promise in treating various diseases, as exemplified by the use of chimeric antigen receptor (CAR) T cells as cancer therapy. However, most CAR designs lack computation features and cannot reprogram multiple immune cell types in a coordinated manner. Here, leveraging our split, universal, and programmable (SUPRA) CAR system, we develop an inhibitory feature, achieving a three-input logic, and demonstrate that this programmable system is functional in diverse adaptive and innate immune cells. We also create an inducible multi-cellular NIMPLY circuit, kill switch, and a synthetic intercellular communication channel. Our work highlights that a simple split CAR design can generate diverse and complex phenotypes and provide a foundation for engineering an immune cell consortium with user-defined functionalities.

In recent years, chimeric antigen receptor (CAR) T cell cancer immunotherapies have advanced substantially in the clinic. However, challenges related to safety persist; one major concern occurs when CARs trigger a response to antigen present on healthy cells (on-target, off-tumor response). A strategy to ameliorate this relies on the complex relationship between receptor affinity and signaling, such that one can engineer a CAR that is only activated by tumor cells expressing high antigen levels. Here, we developed a CAR T cell display platform with stable genomic expression and rapid functional screening based on interleukin-2 signaling. Starting with a CAR with high affinity toward its target antigen, we combined CRISPR-Cas9 genome editing and deep mutational scanning to generate a library of antigen-binding domain variants. This library was subjected to multiple rounds of selection based on either antigen binding or cell signaling. Deep sequencing of the resulting libraries and a comparative analysis revealed the enrichment and depletion of specific variants from which we selected CARs that were selectively activated by tumor cells based on antigen expression levels. Our platform demonstrates how directed evolution based on functional screening and deep sequencing-guided selection can be combined to enhance the selectivity and safety of CARs., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

T-cell acute lymphoblastic leukemia (T-ALL), a T-cell malignant disease that mainly affects children, is still a medical challenge, especially for refractory patients for whom therapeutic options are scarce. Recent advances in immunotherapy for B-cell malignancies based on increasingly efficacious monoclonal antibodies (mAbs) and chimeric antigen receptors (CARs) have been encouraging for non-responding or relapsing patients suffering from other aggressive cancers like T-ALL. However, secondary life-threatening T-cell immunodeficiency due to shared expression of targeted antigens by healthy and malignant T cells is a main drawback of mAb-or CAR-based immunotherapies for T-ALL and other T-cell malignancies. This review provides a comprehensive update on the different immunotherapeutic strategies that are being currently applied to T-ALL. We highlight recent progress on the identification of new potential targets showing promising preclinical results and discuss current challenges and opportunities for developing novel safe and efficacious immunotherapies for T-ALL.

The impressive success of chimeric antigen receptor (CAR)-T cell therapies in treating advanced B-cell malignancies has spurred a frenzy of activity aimed at developing CAR-T therapies for other cancers, particularly solid tumors, and optimizing engineered T cells for maximum clinical benefit in many different disease contexts. A rapidly growing body of design work is examining every modular component of traditional single-chain CARs as well as expanding out into many new and innovative engineered immunoreceptor designs that depart from this template. New approaches to immune cell and receptor engineering are being reported with rapidly increasing frequency, and many recent high-quality reviews (including one in this special issue) provide comprehensive coverage of the history and current state of the art in CAR-T and related cellular immunotherapies. In this review, we step back to examine our current understanding of the structure-function relationships in natural and engineered lymphocyte-activating receptors, with an eye towards evaluating how well the current-generation CAR designs recapitulate the most desirable features of their natural counterparts. We identify key areas that we believe are under-studied and therefore represent opportunities to further improve our grasp of form and function in natural and engineered receptors and to rationally design better therapeutics.

Immune checkpoint inhibitors have redefined the treatment of cancer, but their efficacy depends critically on the presence of sufficient tumor-specific lymphocytes, and cellular immunotherapies develop rapidly to fill this gap. The paucity of suitable extracellular and tumor-associated antigens in solid cancers necessitates the use of neoantigen-directed T-cell-receptor (TCR)-engineered cells, while prevention of tumor evasion requires combined targeting of multiple neoepitopes. These can be currently identified within 2 weeks by combining cutting-edge next-generation sequencing with bioinformatic pipelines and used to select tumor-reactive TCRs in a high-throughput manner for expeditious scalable non-viral gene editing of autologous or allogeneic lymphocytes. "Young" cells with a naive, memory stem or central memory phenotype can be additionally armored with "next-generation" features against exhaustion and the immunosuppressive tumor microenvironment, where they wander after reinfusion to attack heavily pretreated and hitherto hopeless neoplasms. Facilitated by major technological breakthroughs in critical manufacturing steps, based on a solid preclinical rationale, and backed by rapidly accumulating evidence, TCR therapies break one bottleneck after the other and hold the promise to become the next immuno-oncological revolution.

The successful implementation of chimeric antigen receptor (CAR)-T cell therapy in the clinical context of B cell malignancies has paved the way for further development in the more critical setting of acute myeloid leukemia (AML). Among the potentially targetable AML antigens, CD33 is insofar one of the main validated molecules. Here, we describe the feasibility of engineering cytokine-induced killer (CIK) cells with a CD33.CAR by using the latest optimized version of the non-viral Sleeping Beauty (SB) transposon system "SB100X-pT4." This offers the advantage of improving CAR expression on CIK cells, while reducing the amount of DNA transposase as compared to the previously employed "SB11-pT" version. SB-modified CD33.CAR-CIK cells exhibited significant antileukemic activity in vitro and in vivo in patient-derived AML xenograft models, reducing AML development when administered as an "early treatment" and delaying AML progression in mice with established disease. Notably, by exploiting an already optimized xenograft chemotherapy model that mimics human induction therapy in mice, we demonstrated for the first time that CD33.CAR-CIK cells are also effective toward chemotherapy resistant/residual AML cells, further supporting its future clinical development and implementation within the current standard regimens., (Copyright © 2020 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.)

Most studies on genetic engineering technologies for cancer immunotherapy based on allogeneic donors have focused on adaptive immunity. However, the main limitation of such approaches is that they can lead to severe graft-versus-host disease (GvHD). An alternative approach would bolster innate immunity by relying on the natural tropism of some subsets of the innate immune system, such as γδ T and natural killer (NK) cells, for the tumor microenvironment and their ability to kill in a major histocompatibility complex (MHC)-independent manner. γδ T and NK cells have the unique ability to bridge innate and adaptive immunity while responding to a broad range of tumors. Considering these properties, γδ T and NK cells represent ideal sources for developing allogeneic cell therapies. Recently, significant efforts have been made to exploit the intrinsic anti-tumor capacity of these cells for treating hematologic and solid malignancies using genetic engineering approaches such as chimeric antigen receptor (CAR) and T cell receptor (TCR). Here, we review over 30 studies on these two approaches that use γδ T and NK cells in adoptive cell therapy (ACT) for treating cancer. Based on those studies, we propose several promising strategies to optimize the clinical translation of these approaches.

Chimeric antigen receptor modified T cells (CAR-T) have yielded impressive clinical outcomes in treating hematopoietic malignancies. However, relapses have occurred in a substantial number of patients and limited the development of CAR-T therapy. Most underlying reasons for these relapses can be attributed to poor persistence and rapid exhaustion of CAR-T cells in vivo . Despite multiple strategies having been developed, how to improve CAR-T persistence or resist exhaustion while maintaining sufficient cytotoxic functions is still a great challenge. Here we discuss engineering cytoplasmic signaling as an important strategy for CAR optimization. This review summarizes recent advances showing that the anti-tumor function of CAR-T cells can be improved by optimizing the CD3ζ domain or downstream signaling of CD28ζ CAR., (Copyright © 2020 Meng, Jing, Qian, Zhou and Sun.)

Background: Acute myeloid leukemia (AML) is a hematopoietic malignancy which is biologically, phenotypically and genetically very heterogeneous. Outcome of patients with AML remains dismal, highlighting the need for improved, less toxic therapies. Chimeric antigen receptor T-cell (CART) immunotherapies for patients with refractory or relapse (R/R) AML are challenging because of the absence of a universal pan-AML target antigen and the shared expression of target antigens with normal hematopoietic stem/progenitor cells (HSPCs), which may lead to life-threating on-target/off-tumor cytotoxicity. CD33-redirected and CD123-redirected CARTs for AML are in advanced preclinical and clinical development, and they exhibit robust antileukemic activity. However, preclinical and clinical controversy exists on whether such CARTs are myeloablative., Methods: We set out to comparatively characterize in vitro and in vivo the efficacy and safety of 41BB-based and CD28-based CARCD123. We analyzed 97 diagnostic and relapse AML primary samples to investigate whether CD123 is a suitable immunotherapeutic target, and we used several xenograft models and in vitro assays to assess the myeloablative potential of our second-generation CD123 CARTs., Results: Here, we show that CD123 represents a bona fide target for AML and show that both 41BB-based and CD28-based CD123 CARTs are very efficient in eliminating both AML cell lines and primary cells in vitro and in vivo. However, both 41BB-based and CD28-based CD123 CARTs ablate normal human hematopoiesis and prevent the establishment of de novo hematopoietic reconstitution by targeting both immature and myeloid HSPCs., Conclusions: This study calls for caution when clinically implementing CD123 CARTs, encouraging its preferential use as a bridge to allo-HSCT in patients with R/R AML., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2020. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Multiple myeloma (MM) is a haematologic malignancy with significant improvements in the overall survival over the last decade. However, patients still relapse and die due to a lack of treatment options. Ultimately, novel therapies with the potential for long term remissions are needed for patients with advanced MM. Research efforts for such immune therapies were not successful until recently when the first immunotherapies for MM were approved in 2015 and many more are under development. In this review, we focus on adoptive cell therapies including CAR T-cell and CAR NK-cell therapies for patients with MM. We will provide an update on clinical and translational advances with a focus on results from ongoing clinical trials with BCMA targeted cellular therapies and the development of other novel targets, changes in the manufacturing process, trials focusing on earlier lines of therapy and combinations with other therapies as well as off the shelf products., Competing Interests: Declaration of competing interest Urvi Shah has received research support from Celgene and honoraria from Physicians' Education Resource. Sham Mailankody has received grant support from National Cancer Institute, research support from Takeda Oncology, Juno/Celgene, Janssen and Allogene Therapeutics and honoraria from Physicians’ Education Resource., (Copyright © 2020. Published by Elsevier Ltd.)

The anti-tumor efficacy of TCR-engineered T cells in vivo depends largely on less-differentiated subsets such as T cells with naïve-like T cell (T N ) phenotypes with greater expansion and long-term persistence. To increase these subsets, we compared the generation of New York esophageal squamous cell carcinoma-1 (NY-ESO-1)-specific T cells under supplementation with either IL-2 or IL-7/IL-15. PBMCs were transduced with MS3II-NY-ESO-1-siTCR retroviral vector. T cell generation was adapted from a CD19-specific CART cell production protocol. Comparable results in viability, expansion and transduction efficiency of T cells under stimulation with either IL-2 or IL-7/IL-15 were observed. IL-7/IL-15 led to an increase of CD4 + T cells and a decrease of CD8 + T cells, enriched the amount of T N among CD4 + T cells but not among CD8 + T cells. In a 51 Cr release assay, similar specific lysis of NY-ESO-1-positive SW982 sarcoma cells was achieved. However, intracellular cytokine staining revealed a significantly increased production of IFN-γ and TNF-α in T cells generated by IL-2 stimulation. To validate these unexpected findings, NY-ESO-1-specific T cell production was evaluated in another protocol originally established for TCR-engineered T cells. IL-7/IL-15 increased the proportion of T N . However, the absolute number of T N did not increase due to a significantly slower expansion of T cells with IL-7/IL-15. In conclusion, IL-7/IL-15 does not seem to be superior to IL-2 for the generation of NY-ESO-1-specific T cells. This is in sharp contrast to the observations in CD19-specific CART cells. Changes of cytokine cocktails should be carefully evaluated for individual vector systems.

Although chimeric antigen receptor (CAR) T cell therapies have demonstrated considerable success in treating hematologic malignancies, they have simultaneously been plagued by a cytokine release syndrome (CRS) that can harm or even kill the cancer patient. We describe a CAR T cell strategy in which CAR T cell activation and cancer cell killing can be sensitively regulated by adjusting the dose of a low molecular weight adapter that must bridge between the CAR T cell and cancer cell to initiate tumor eradication. By controlling the concentration and dosing schedule of adapter administration, we document two methods that can rapidly terminate (<3 h) a pre-existing CRS-like toxicity and two unrelated methods that can pre-emptively prevent a CRS-like toxicity that would have otherwise occurred. Because all four methods concurrently enhance CAR T cell potency, we conclude that proper use of bispecific adapters could potentially avoid a life-threatening CRS while enhancing CAR T cell tumoricidal activity.

Cancer therapy has entered a new era, transitioning from unspecific chemotherapeutic agents to increasingly specific immune-based therapeutic strategies. Among these, chimeric antigen receptor (CAR) T cells have shown unparalleled therapeutic potential in treating refractory hematological malignancies. In contrast, solid tumors pose a much greater challenge to CAR T cell therapy, which has yet to be overcome. As this novel therapeutic modality matures, increasing effort is being invested to determine the optimal structure and properties of CARs to facilitate the transition from empirical testing to the rational design of CAR T cells. In this review, we highlight how individual CAR domains contribute to the success and failure of this promising treatment modality and provide an insight into the most notable advances in the field of CAR T cell engineering.

Chronic myeloid leukemia (CML) is a chronic disease resulting in myeloid cell expansion through expression of the BCR-ABL1 fusion transcript. Tyrosine kinase inhibitors (TKI) have significantly increased survival of patients with CML, and deep responders may consider stopping the treatment. However, more than 50% of patients relapse and restart TKI, subsequently suffering unknown toxicity. Because CML is a model immune system-sensitive disease, we hypothesize that chimeric antigen receptor (CAR) T cells targeting IL1 receptor-associated protein (IL1RAP) in quiescent CML stem cells may offer an opportunity for a permanent cure. In this study, we produced and molecularly characterized a specific monoclonal anti-IL1RAP antibody from which fragment antigen-binding nucleotide coding sequences were cloned as a single chain into a lentiviral backbone and secured with the suicide gene iCASP9/rimiducid system. Our CAR T-cell therapy exhibited cytotoxicity against both leukemic stem cells and, to a lesser extent, monocytes expressing IL1RAP, with no apparent effect on the hematopoietic system, including CD34 + stem cells. This suggests IL1RAP as a tumor-associated antigen for immunotherapy cell targeting. IL1RAP CAR T cells were activated in the presence of IL1RAP + cell lines or primary CML cells, resulting in secretion of proinflammatory cytokines and specifically killing in vitro and in a xenograft murine model. Overall, we demonstrate the proof of concept of a CAR T-cell immunotherapy approach in the context of CML that is applicable for young patients and primary TKI-resistant, intolerant, or allograft candidate patients. SIGNIFICANCE: These findings present the first characterization and proof of concept of a chimeric antigen receptor directed against IL1RAP expressed by leukemic stem cells in the context of CML., (©2018 American Association for Cancer Research.)

Most solid tumors are comprised of multiple clones that express orthogonal antigens, suggesting that novel strategies must be developed in order to adapt chimeric antigen receptor (CAR) T-cell therapies to treat heterogeneous solid tumors. Here, we utilized a cocktail of low-molecular-weight bispecific adapters, each comprised of fluorescein linked to a different tumor-specific ligand, to bridge between an antifluorescein CAR on the engineered T cell and a unique antigen on the cancer cell. This formation of an immunologic synapse between the CAR T cell and cancer cell enabled use of a single antifluorescein CAR T cell to eradicate a diversity of antigenically different solid tumors implanted concurrently in NSG mice. Based on these data, we suggest that a carefully designed cocktail of bispecific adapters in combination with antifluorescein CAR T cells can overcome tumor antigen escape mechanisms that lead to disease recurrence following many CAR T-cell therapies. SIGNIFICANCE: A cocktail of tumor-targeted bispecific adapters greatly augments CAR T-cell therapies against heterogeneous tumors, highlighting its potential for broader applicability against cancers where standard CAR T-cell therapy has failed., (©2018 American Association for Cancer Research.)

T cells engineered with chimeric antigen receptors (CARs) are emerging as powerful cancer immunotherapies. Remarkable efficacies have been demonstrated in treating B-cell malignancies with CAR-T cells, leading to the FDA's first approval of gene therapy. Currently, numerous clinical trials for hematological malignancies and solid tumors are underway worldwide. Production of CAR-T cells with proper qualities is essential for CAR-T success in vivo. Here we detail optimized protocols for the generation of CAR-T cells for preclinical studies using lentiviral gene transfer, expansion of CAR-T cells in culture, detection of CAR expression, and evaluation of CAR-T cellular cytotoxicity in vitro.

Recent outstanding clinical results produced by engineered T cells, including chimeric antigen receptors, have already facilitated further research that broadens their applicability. One such direction is to explore new T cell sources for allogeneic "off-the-shelf" adoptive immunotherapy. Human pluripotent stem cells could serve as an alternative cell source for this purpose due to their unique features of infinite propagation ability and pluripotency. Here, we describe the current state of engineered T cell transfer with the focus on cell manufacturing processes and the potentials and challenges of induced pluripotent stem cell-derived T cells as a starting material to construct off-the-shelf T-cell banks., (© 2018 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.)

Introduction: Cancer therapy has been transformed by the demonstration that tumor-specific T-cells can eliminate tumor cells in a clinical setting with minimal long-term toxicity. However, significant success in the treatment of leukemia and lymphoma with T-cells using native receptors or redirected with chimeric antigen receptors (CARs) has not been recapitulated in the treatment of solid tumors. This lack of success is likely related to the paucity of costimulatory and cytokine signaling available in solid tumors, in addition to a range of inhibitory mechanisms., Areas Covered: We summarize the latest developments in engineered T-cell immunotherapy, describe the limitations of these approaches in treating solid tumors, and finally highlight several strategies that may be useful in mediating solid tumor responses in the future, while also ensuring safety of engineered cells., Expert Opinion: CAR-T therapies require further engineering to achieve their potential against solid tumors. Facilitating cytokine signaling in CAR T-cells appears to be essential in achieving better responses. However, the engineering of T-cells with potentially unchecked proliferation and potency raises the question of whether the simultaneous combination of enhancements will prove safe, necessitating continued advancements in regulating CAR-T activity at the tumor site and methods to safely switch off these engineered cells.

T cells genetically engineered with tumor antigen-specific T-cell receptor (TCR) genes have demonstrated therapeutic potential in patients with solid tumors. In order to achieve broader application, an efficient method to identify TCR genes for an array of tumor antigens and HLA restriction elements is required. Here, we have developed a method to construct a TCR-expression library from specimens, including frozen tumor biopsies, that contain antigen-specific T cells. TCR-expressing cassettes were constructed and cloned in a retroviral plasmid vector within 24 hours by unbiased PCR amplification of TCR α and β chain variable regions assembled with TCR constant regions. The method was validated by constructing TCR-expressing vectors from tumor antigen-specific T-cell clones and functionally assessing TCR gene-transduced T cells. We applied this method to frozen ovarian tumor specimens that were infiltrated by tumor antigen-specific T cells. The tumor-derived TCR libraries were expressed in peripheral T cells from healthy volunteers and screened for tumor antigen-specific TCR pairs with the use of an MHC/peptide tetramer reagent. Tumor antigen-specific TCR-expressing transgenes were recovered from isolated tetramer-positive T cells. Peripheral T cells that were engineered with library-derived TCR gene showed potent therapeutic antitumor effect in a tumor xenograft model. Our method can efficiently and rapidly provide tumor-specific TCR-expressing viral vectors for the manufacture of therapeutic and personalized antitumor T-cell products. Cancer Immunol Res; 6(5); 594-604. ©2018 AACR ., (©2018 American Association for Cancer Research.)

Adoptive T cell transfer (ACT) is a new area of transfusion medicine involving the infusion of lymphocytes to mediate antitumor, antiviral, or anti-inflammatory effects. The field has rapidly advanced from a promising form of immuno-oncology in preclinical models to the recent commercial approvals of chimeric antigen receptor (CAR) T cells to treat leukemia and lymphoma. This Review describes opportunities and challenges for entering mainstream oncology that presently face the CAR T field, with a focus on the challenges that have emerged over the past several years., (Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Interleukin-2 (IL-2) is a cytokine required for effector T cell expansion, survival, and function, especially for engineered T cells in adoptive cell immunotherapy, but its pleiotropy leads to simultaneous stimulation and suppression of immune responses as well as systemic toxicity, limiting its therapeutic use. We engineered IL-2 cytokine-receptor orthogonal ( ortho ) pairs that interact with one another, transmitting native IL-2 signals, but do not interact with their natural cytokine and receptor counterparts. Introduction of ortho IL-2Rβ into T cells enabled the selective cellular targeting of ortho IL-2 to engineered CD4 + and CD8 + T cells in vitro and in vivo, with limited off-target effects and negligible toxicity. Ortho IL-2 pairs were efficacious in a preclinical mouse cancer model of adoptive cell therapy and may therefore represent a synthetic approach to achieving selective potentiation of engineered cells., (Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Recent advances in adoptive immunotherapy using cytotoxic T lymphocytes (CTLs) have led to moderate therapeutic anti-cancer effects in clinical trials. However, a critical issue, namely that CTLs collected from patients are easily exhausted during expansion culture, has yet to be solved. To address this issue, we have been developing a strategy which utilizes induced pluripotent stem cell (iPSC) technology. This strategy is based on the idea that when iPSCs are produced from antigen-specific CTLs, CTLs regenerated from such iPSCs should show the same antigen specificity as the original CTLs. Pursuing this idea, we previously succeeded in regenerating melanoma antigen MART1-specific CTLs, and more recently in producing potent CTLs expressing CD8αβ heterodimer. We are now developing a novel method by which non-T derived iPSCs are transduced with exogenous T cell receptor genes. If this method is applied to Human Leukocyte Antigen (HLA) haplotype-homozygous iPSC stock, it will be possible to prepare "off-the-shelf" T cells. As a first-in-human trial, we are planning to apply our strategy to relapsed acute myeloid leukemia patients by targeting the WT1 antigen.

Chimeric antigen receptor (CAR) T-cell technology has seen a rapid development over the last decade mostly due to the potential that these cells may have in treating malignant diseases. It is a generally accepted principle that very few therapeutic compounds deliver a clinical response without treatment-related toxicity, and studies have shown that CAR T-cells are not an exception to this rule. While large multinational drug companies are currently investigating the potential role of CAR T-cells in hematological oncology, the potential of such cellular therapies are being recognized worldwide as they are expected to expand in the patient to support the establishment of the immune memory, provide a continuous surveillance to prevent and/or treat a relapse, and keep the targeted malignant cell subpopulation in check. In this article, we present the possible advantages of using CAR T-cells in treating acute lymphoblastic leukemia, presenting the technology and the current knowledge in their preclinical and early clinical trial use. Thus, this article first presents the main present-day knowledge on the standard of care for acute lymphoblastic leukemia. Afterward, current knowledge is presented about the use of CAR T-cells in cancer immunotherapy, describing their design, the molecular constructs, and the preclinical data on murine models to properly explain the background for their clinical use. Last, but certainly not least, this article presents the use of CAR T-cells for the immunotherapy of B-cell acute lymphoblastic leukemia, describing both their potential clinical advantages and the possible side effects.

Hemophilia A is a bleeding disorder caused by mutations in the gene encoding factor VIII (FVIII), a cofactor protein that is essential for normal blood clotting. Approximately, 1 in 3 patients with severe hemophilia A produce neutralizing antibodies (inhibitors) that block its biologic function in the clotting cascade. Current efforts to eliminate inhibitors consist of repeated FVIII injections under what is termed an "ITI" protocol (Immune Tolerance Induction). However, this method is extremely costly and approximately 30% of patients undergoing ITI do not achieve peripheral tolerance. Human T regulatory cells (Tregs) have been proposed as a new strategy to treat this antidrug antibody response, as well as other diseases. Polyclonal Tregs are nonspecific and could potentially cause general immunosuppression. Novel approaches to induce tolerance to FVIII include the use of engineered human and mouse antigen-specific Tregs, or alternatively antigen-specific cytotoxic cells, to delete, anergize, or kill FVIII-specific lymphocytes. In this review, we discuss the current state of engineered T-cell therapies, and we describe the recent progress in applying these therapies to induce FVIII-specific tolerance., (Published by Elsevier Inc.)

Background: Major barriers in using classical FOXP3+ regulatory T cells (Tregs) in clinical practice are their low numbers in the circulation, the lack of specific cell surface markers for efficient purification and the loss of expression of Treg signature molecules and suppressive function after in vitro expansion or in a pro-inflammatory microenviroment. A surface molecule with potent immunosuppressive function is the human leukocyte antigen-G (HLA-G), which is normally expressed in placenta protecting the "semi-allogeneic" fetus from maternal immune attack. Because HLA-G expression is strongly regulated by methylation, we asked whether hypomethylating agents (HA) may be used in vitro to induce HLA-G expression on conventional T cells and convert them to Tregs., Methods: Human peripheral blood T cells were exposed to azacytidine/decitabine and analyzed for HLA-G expression and their in vitro suppressor properties., Results: HA treatment induces de novo expression of HLA-G on T cells through hypomethylation of the HLA-G proximal promoter. The HA-induced CD4 + HLA-G pos T cells are FOXP3 negative and have potent in vitro suppression function, which is dependent to a large extent, but not exclusively, on the HLA-G molecule. Converted HLA-G pos suppressors retain their suppressor function in the presence of tumor necrosis factor (TNF) and preserve hypomethylated the HLA-G promoter for at least 2 days after azacytidine exposure. Decitabine-treated T cells suppressed ex vivo the proliferation of T cells isolated from patients suffering from graft-versus-host disease (GVHD)., Discussion: We propose, in vitro generation of HLA-G-expressing T cells through pharmacological hypomethylation as a simple, Good Manufacturing Practice (GMP)-compatible and efficient strategy to produce a stable Treg subset of a defined phenotype that can be easily purified for adoptive immunotherapy., (Copyright © 2017 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.)

Immunotherapy with T cell modified with gamma-retroviral or lentiviral (LV) vectors to express a chimeric antigen receptor (CAR) has shown remarkable efficacy in clinical trials. However, the potential for insertional mutagenesis and genotoxicity of viral vectors is a safety concern, and their cost and regulatory demands a roadblock for rapid and broad clinical translation. Here, we demonstrate that CAR T cells can be engineered through non-viral Sleeping Beauty (SB) transposition of CAR genes from minimalistic DNA vectors called minicircles (MCs). We analyzed genomic distribution of SB and LV integrations and show that a significantly higher proportion of MC-derived CAR transposons compared with LV integrants had occurred outside of highly expressed and cancer-related genes into genomic safe harbor loci that are not expected to cause mutagenesis or genotoxicity. CD19-CAR T cells engineered with our enhanced SB approach conferred potent reactivity in vitro and eradicated lymphoma in a xenograft model in vivo. Intriguingly, electroporation of SB MCs is substantially more effective and less toxic compared with conventional plasmids, and enables cost-effective rapid preparation of therapeutic CAR T-cell doses. This approach sets a new standard in advanced cellular and gene therapy and will accelerate and increase the availability of CAR T-cell therapy to treat hematologic malignancies.

Adoptive transfer of genetically engineered T cells can lead to profound and durable responses in patients with hematologic malignancies, generating enormous enthusiasm among scientists, clinicians, patients, and biotechnology companies. The success of adoptive cellular immunotherapy depends upon the ability to manufacture good quality T cells. We discuss here the methodologies and reagents that are used to generate T cells for the preclinical study of chimeric antigen receptor T cell therapy for acute myeloid leukemia (AML).

Pre-emptive cancer immunotherapy by donor lymphocyte infusion (DLI) using cytokine-induced killer (CIK) cells may be beneficial to prevent relapse with a reduced risk of causing graft-versus-host-disease. CIK cells are a heterogeneous effector cell population including T cells (CD3(+) CD56(-) ), natural killer (NK) cells (CD3(-) CD56(+) ) and natural killer T (T-NK) cells (CD3(+) CD56(+) ) that exhibit non-major histocompatibility complex (MHC)-restricted cytotoxicity and are generated by ex vivo expansion of peripheral blood mononuclear cells in the presence of interferon (IFN)-γ, anti-CD3 antibody, interleukin-2 (IL-2) and interleukin-15 (IL-15). To facilitate selective target-cell recognition and enhance specific cytotoxicity against B-cell acute lymphoblastic leukemia (B-ALL), we transduced CIK cells with a lentiviral vector encoding a chimeric antigen receptor (CAR) that carries a composite CD28-CD3ζ domain for signaling and a CD19-specific scFv antibody fragment for cell binding (CAR 63.28.z). In vitro analysis revealed high and specific cell killing activity of CD19-targeted CIK/63.28.z cells against otherwise CIK-resistant cancer cell lines and primary B-ALL blasts, which was dependent on CD19 expression and CAR signaling. In a xenograft model in immunodeficient mice, treatment with CIK/63.28.z cells in contrast to therapy with unmodified CIK cells resulted in complete and durable molecular remissions of established primary pre-B-ALL. Our results demonstrate potent antileukemic activity of CAR-engineered CIK cells in vitro and in vivo, and suggest this strategy as a promising approach for adoptive immunotherapy of refractory pre-B-ALL., (© 2016 UICC.)

Novel cell therapies derived from human T lymphocytes are exhibiting enormous potential in early-phase clinical trials in patients with hematologic malignancies. Ex vivo modification of T cells is currently limited to a small number of centers with the required infrastructure and expertise. The process requires isolation, activation, transduction, expansion and cryopreservation steps. To simplify procedures and widen applicability for clinical therapies, automation of these procedures is being developed. The CliniMACS Prodigy (Miltenyi Biotec) has recently been adapted for lentiviral transduction of T cells and here we analyse the feasibility of a clinically compliant T-cell engineering process for the manufacture of T cells encoding chimeric antigen receptors (CAR) for CD19 (CAR19), a widely targeted antigen in B-cell malignancies. Using a closed, single-use tubing set we processed mononuclear cells from fresh or frozen leukapheresis harvests collected from healthy volunteer donors. Cells were phenotyped and subjected to automated processing and activation using TransAct, a polymeric nanomatrix activation reagent incorporating CD3/CD28-specific antibodies. Cells were then transduced and expanded in the CentriCult-Unit of the tubing set, under stabilized culture conditions with automated feeding and media exchange. The process was continuously monitored to determine kinetics of expansion, transduction efficiency and phenotype of the engineered cells in comparison with small-scale transductions run in parallel. We found that transduction efficiencies, phenotype and function of CAR19 T cells were comparable with existing procedures and overall T-cell yields sufficient for anticipated therapeutic dosing. The automation of closed-system T-cell engineering should improve dissemination of emerging immunotherapies and greatly widen applicability., (Copyright © 2016. Published by Elsevier Inc.)

The field of adoptive cell transfer includes chimeric antigen receptor (CAR) engineered T cells, constructs that emerged from basic research into principles of immunology and have transformed into clinically effective therapies for haematological malignancies. T cells engineered to express these artificial receptors hold great promise, but also carry significant risk. While permanent genetic modification of mature T cells appears safe, modulating their in vivo function is difficult, partly because the robust response can trigger other arms of the immune system. Suicide systems and toxicity management with cytokine blockade or signal transduction modulators have emerged as a new frontier in this field, a far cry from early problems getting CAR T cells to work at all. Currently, clinical trials in patients with relapsed or refractory B cell malignancies treated with CD19-specific CAR T cells have induced durable remissions in adults and children. Results from these trials indicate that more work needs to be done to understand biomarkers of efficacy, the role of T cell persistence and how to integrate this care into standard practice. Cell therapy will not be a 'one size fits all' class of medicine, and here we will discuss the development of this therapy and important questions for its future., (© 2015 John Wiley & Sons Ltd.)

Tumors originate from a number of genetic events that deregulate homeostatic mechanisms controlling normal cell behavior. The immune system, devoted to patrol the organism against pathogenic events, can identify transformed cells, and in several cases cause their elimination. It is however clear that several mechanisms encompassing both central and peripheral tolerance limit antitumor immunity, often resulting into progressive diseases. Adoptive T-cell therapy with either allogeneic or autologous T cells can transfer therapeutic immunity. To date, genetic engineering of T cells appears to be a powerful tool for shaping tumor immunity. In this review, we discuss the most recent achievements in the areas of suicide gene therapy, and TCR-modified T cells and chimeric antigen receptor gene-modified T cells. We provide an overview of current strategies aimed at improving the safety and efficacy of these approaches, with an outlook on prospective developments., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

It is becoming increasingly clear that adoptive immunotherapy with genetically engineered T cells has the potential to control and even cure cancer in some patients. On the other hand, severe adverse events associated with efficacy have frequently been reported in clinical trials. Current and near-future challenges for the development of adoptive immunotherapy of cancer using genetically engineered T cells include minimization and prediction of adverse events; identification of new and effective targets, including patient-specific mutations; improvement in T cell functionality, persistence, and memory formation capacity; and utilization of allogeneic or cell line-based T cells.

The promising clinical results obtained with engineered T cells, including chimeric antigen receptor (CAR) therapy, call for further advancements to facilitate and broaden their applicability. One potentially beneficial innovation is to exploit new T cell sources that reduce the need for autologous cell manufacturing and enable cell transfer across histocompatibility barriers. Here we review emerging T cell engineering approaches that utilize alternative T cell sources, which include virus-specific or T cell receptor-less allogeneic T cells, expanded lymphoid progenitors, and induced pluripotent stem cell (iPSC)-derived T lymphocytes. The latter offer the prospect for true off-the-shelf, genetically enhanced, histocompatible cell therapy products., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Natural killer (NK) cells can kill transformed cells and represent a promising tool for the treatment of cancer. Their function is governed by a balance of stimulatory and inhibitory signals triggered by surface receptors. Advances in NK cell therapy require the development of dependable methods for obtaining an adequate number of effector cells; additional activation or genetic modification may further increase their anticancer capacity. A method for NK cell expansion used in our laboratory relies on a genetically modified form of the K562 myeloid leukemia cell line, engineered to express a membrane-bound form of interleukin-15 and the ligand for the costimulatory molecule 4-1BB (CD137). Expanded NK cells can be transduced with genes encoding chimeric antigen receptors that stimulate tumor cell-specific cytotoxicity. These methods for NK cell expansion and genetic modification have been adapted to large-scale, clinical-grade, Current Good Manufacturing Practice conditions and support two active clinical trials. Summarized are current efforts for NK cell immunotherapy for cancer and future perspectives., (© 2011 John Wiley & Sons A/S.)