ABSTRACT
Stem cell-based therapies offer promising avenues for treating inflammatory diseases owing to their immunomodulatory properties. However, challenges persist regarding their survival and efficacy in inflamed tissues. Our study introduces a novel approach by engineering adipose-derived stem cells (ADSCs) to enhance their viability in inflammatory environments and boost the secretion of paracrine factors for treating inflammatory bowel disease (IBD). An arginine-glycine-aspartate peptide-poly (ethylene glycol)-chlorin e6 conjugate (RPC) was synthesized and coupled with ADSCs, resulting in RPC-labeled ADSCs (ARPC). This conjugation strategy employed RGD-integrin interaction to shield stem cells and allowed visualization and tracking using chlorin e6. The engineered ARPC demonstrated enhanced viability and secretion of paracrine factors upon light irradiation, regulating the inflammatory microenvironment. RNA-sequencing analysis unveiled pathways favoring angiogenesis, DNA repair, and exosome secretion in ARPC(+) while downregulating inflammatory pathways. In in vivo models of acute and chronic IBD, ARPC(+) treatment led to reduced inflammation, preserved colon structure, and increased populations of regulatory T cells, highlighting its therapeutic potential. ARPC(+) selectively homed to inflammatory sites, demonstrating its targeted effect. Overall, ARPC(+) exhibits promise as an effective and safe therapeutic strategy for managing inflammatory diseases like IBD by modulating immune responses and creating an anti-inflammatory microenvironment.
ABSTRACT
Efficient and nontoxic delivery of foreign cargo into cells is a critical step in many biological studies and cell engineering workflows with applications in areas such as biomanufacturing and cell-based therapeutics. However, effective molecular delivery into cells involves optimizing several experimental parameters. In the case of electroporation-based intracellular delivery, there is a need to optimize parameters like pulse voltage, duration, buffer type, and cargo concentration for each unique application. Here, we present the protocol for fabricating and utilizing a high-throughput multi-well localized electroporation device (LEPD) assisted by deep learning-based image analysis to enable rapid optimization of experimental parameters for efficient and nontoxic molecular delivery into cells. The LEPD and the optimization workflow presented herein are relevant to both adherent and suspended cell types and different molecular cargo (DNA, RNA, and proteins). The workflow enables multiplexed combinatorial experiments and can be adapted to cell engineering applications requiring in vitro delivery. Key features ⢠A high-throughput multi-well localized electroporation device (LEPD) that can be optimized for both adherent and suspended cell types. ⢠Allows for multiplexed experiments combined with tailored pulse voltage, duration, buffer type, and cargo concentration. ⢠Compatible with various molecular cargoes, including DNA, RNA, and proteins, enhancing its versatility for cell engineering applications. ⢠Integration with deep learning-based image analysis enables rapid optimization of experimental parameters.
ABSTRACT
Chimeric antigen receptor (CAR) is a synthetic receptor that induces T cell-mediated lysis of abnormal cells. As cancer driver proteins are present at low levels on the cell surface, they can cause weak CAR reactivity, resulting in antigen sensitivity defects and consequently limited therapeutic efficacy. Although affinity maturation enhances the efficacy of CAR-T cell therapy, it causes off-target cross-reactions resulting in adverse effects. Preferentially expressed antigen in melanoma (PRAME) is an intracellular oncoprotein that is overexpressed in various tumors and restricted in normal tissues, except the testis. Therefore, PRAME could be an ideal target for cancer immunotherapy. In this study, we developed an experimental CAR system comprising six single-chain variable fragments that specifically recognizes the PRAMEp301/HLA-A∗24:02 complex. Cell-mediated cytotoxicity was demonstrated using a panel of CARs with a wide range of affinities (KD = 10-10-10-7 M) and affinity modulation. CAR-T cells with fast on-rates enhance antigen sensitivity by accelerating the killing rates of these cells. Alanine scanning data demonstrated the potential of genetically engineered CARs to reduce the risk of cross-reactivity, even among CARs with high affinities. Given the correlation between on-rates and dwell time that occurs in rebinding and cell-mediated cytotoxicity, it is proposed that CAR-binding characteristics, including on-rate, play a pivotal role in the lytic capacity of peptide-major histocompatibility complex-targeting CAR-T cells, thus facilitating the development of strategies whereby genetically engineered CARs target intracellular antigens in cancer cells to lyse the cells.
ABSTRACT
Engineering and reprogramming cells has significant therapeutic potential to treat a wide range of diseases, by replacing missing or defective proteins, to provide transcription factors (TFs) to reprogram cell phenotypes, or to provide enzymes such as RNA-guided Cas9 derivatives for CRISPR-based cell engineering. While viral vector-mediated gene transfer has played an important role in this field, the use of mRNA avoids safety concerns associated with the integration of DNA into the host cell genome, making mRNA particularly attractive for in vivo applications. Widespread application of mRNA for cell engineering is limited by its instability in the biological environment and challenges involved in the delivery of mRNA to its target site. In this review, we examine challenges that must be overcome to develop effective therapeutics.
ABSTRACT
Chimeric antigen receptor (CAR) T-cell therapy has proven a breakthrough in cancer treatment in the last decade, giving unprecedented results against hematological malignancies. All approved CAR T-cell products, as well as many being assessed in clinical trials, are generated using viral vectors to deploy the exogenous genetic material into T-cells. Viral vectors have a long-standing clinical history in gene delivery, and thus underwent iterations of optimization to improve their efficiency and safety. Nonetheless, their capacity to integrate semi-randomly into the host genome makes them potentially oncogenic via insertional mutagenesis and dysregulation of key cellular genes. Secondary cancers following CAR T-cell administration appear to be a rare adverse event. However several cases documented in the last few years put the spotlight on this issue, which might have been underestimated so far, given the relatively recent deployment of CAR T-cell therapies. Furthermore, the initial successes obtained in hematological malignancies have not yet been replicated in solid tumors. It is now clear that further enhancements are needed to allow CAR T-cells to increase long-term persistence, overcome exhaustion and cope with the immunosuppressive tumor microenvironment. To this aim, a variety of genomic engineering strategies are under evaluation, most relying on CRISPR/Cas9 or other gene editing technologies. These approaches are liable to introduce unintended, irreversible genomic alterations in the product cells. In the first part of this review, we will discuss the viral and non-viral approaches used for the generation of CAR T-cells, whereas in the second part we will focus on gene editing and non-gene editing T-cell engineering, with particular regard to advantages, limitations, and safety. Finally, we will critically analyze the different gene deployment and genomic engineering combinations, delineating strategies with a superior safety profile for the production of next-generation CAR T-cell.
Subject(s)
Gene Editing , Immunotherapy, Adoptive , Receptors, Chimeric Antigen , T-Lymphocytes , Humans , Immunotherapy, Adoptive/methods , Immunotherapy, Adoptive/adverse effects , Receptors, Chimeric Antigen/genetics , Receptors, Chimeric Antigen/immunology , Gene Editing/methods , T-Lymphocytes/immunology , Animals , Neoplasms/therapy , Neoplasms/immunology , Neoplasms/genetics , Genetic Vectors/genetics , Genetic Vectors/immunology , Genetic Engineering , CRISPR-Cas Systems , Receptors, Antigen, T-Cell/genetics , Receptors, Antigen, T-Cell/immunology , Tumor Microenvironment/immunologyABSTRACT
A range of recombinant monoclonal antibodies (rMAbs) have found application in treating diverse diseases, spanning various cancers and immune system disorders. Chinese hamster ovary (CHO) cells have emerged as the predominant choice for producing these rMAbs due to their robustness, ease of transfection, and capacity for posttranslational modifications akin to those in human cells. Transient transfection and/or stable expression could be conducted to express rMAbs in CHO cells. To bolster the yield of rMAbs in CHO cells, a multitude of approaches have been developed, encompassing vector optimization, medium formulation, cultivation parameters, and cell engineering. This review succinctly outlines these methodologies when also addressing challenges encountered in the production process, such as issues with aggregation and fucosylation.
ABSTRACT
Background: Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) show tremendous promise for cardiac regeneration following myocardial infarction (MI), but their transplantation gives rise to transient ventricular tachycardia (VT) in large-animal MI models, representing a major hurdle to translation. Our group previously reported that these arrhythmias arise from a focal mechanism whereby graft tissue functions as an ectopic pacemaker; therefore, we hypothesized that hPSC-CMs engineered with a dominant negative form of the pacemaker ion channel HCN4 (dnHCN4) would exhibit reduced automaticity and arrhythmogenic risk following transplantation. Methods: We used CRISPR/Cas9-mediated gene-editing to create transgenic dnHCN4 hPSC-CMs, and their electrophysiological behavior was evaluated in vitro by patch-clamp recordings and optical mapping. Next, we transplanted WT and homozygous dnHCN4 hPSC-CMs in a pig MI model and compared post-transplantation outcomes including the incidence of spontaneous arrhythmias and graft structure by immunohistochemistry. Results: In vitro dnHCN4 hPSC-CMs exhibited significantly reduced automaticity and pacemaker funny current (I f ) density relative to wildtype (WT) cardiomyocytes. Following transplantation with either dnHCN4 or WT hPSC-CMs, all recipient hearts showed transmural infarct scar that was partially remuscularized by scattered islands of human myocardium. However, in contrast to our hypothesis, both dnHCN4 and WT hPSC-CM recipients exhibited frequent episodes of ventricular tachycardia (VT). Conclusions: While genetic silencing of the pacemaker ion channel HCN4 suppresses the automaticity of hPSC-CMs in vitro, this intervention is insufficient to reduce VT risk post-transplantation in the pig MI model, implying more complex mechanism(s) are operational in vivo.
ABSTRACT
Automated high-throughput methods that support tracking of mammalian cell growth are currently needed to advance cell line characterization and identification of desired genetic components required for cell engineering. Here, we describe a high-throughput noninvasive assay based on plate reader measurements. The assay relies on the change in absorbance of the pH indicator phenol red. We show that its basic and acidic absorbance profiles can be converted into a cell growth index consistent with cell count profiles, and that, by adopting a computational pipeline and calibration measurements, it is possible to identify a conversion that enables prediction of cell numbers from plate measurements alone. The assay is suitable for growth characterization of both suspension and adherent cell lines when these are grown under different environmental conditions and treated with chemotherapeutic drugs. The method also supports characterization of stably engineered cell lines and identification of desired promoters based on fluorescence output.
Subject(s)
Cell Proliferation , Promoter Regions, Genetic , Animals , Humans , Cell Engineering/methods , Phenolsulfonphthalein , Cell Line , High-Throughput Screening Assays/methods , Cell Culture Techniques/methods , Hydrogen-Ion ConcentrationABSTRACT
Background: CHO cells are preferred for producing biopharmaceuticals, and genome editing technologies offer opportunities to enhance recombinant protein production. Targeting apoptosis-related genes, such as Caspases 8-Associated Protein 2 (CASP8AP2), improves CHO cell viability and productivity. Integrating robust strategies with the CRISPR-Cas9 system enables its application in CHO cell engineering. Objectives: This study was performed to develop a cost-effective protocol using the CRISPR-Cas9 system combined with the HITI strategy for simultaneous CASP8AP2 gene deletion/insertion in CHO cells and to assess its impact on cell viability and protein expression. Materials and Methods: We developed an efficient protocol for CHO cell engineering by combining CRISPR/Cas9 with the HITI strategy. Two distinct sgRNA sequences were designed to target the 3' UTR region of the CASP8AP2 gene using CHOPCHOP software. The gRNAs were cloned into PX459 and PX460-1 vectors and transfected into CHO cells using the cost-effective PEI reagent. A manual selection system was employed to streamline the process of single-cell cloning. MTT assays assessed gene silencing and cell viability at 24, 48, and 72 hours. Flow cytometry evaluated protein expression in CASP8AP2-silenced CHO cells. Results: The study confirmed the robustness of combining CRISPR-Cas9 with the HITI strategy, achieving a high 60% efficiency in generating knockout clones. PEI transfection successfully delivered the constructs to nearly 65% of the clones, with the majority being homozygous. The protocol proved feasible for resource-limited labs, requiring only an inverted fluorescent microscope. CASP8AP2 knockout (CHO-KO) cells exhibited significantly extended cell viability compared to CHO-K1 cells when treated with NaBu, with IC50 values of 7.28 mM and 14.25 mM at 48 hours, respectively (P-value 24 hours ≤ 0.0001, 48 hours ≤ 0.0001, P-value 72 hours = 0.0007). CHO CASP8AP2-silenced cells showed a 1.3-fold increase in JRed expression compared to native cells. Conclusions: CRISPR-Cas9 and HITI strategy was used to efficiently engineer CHO cells for simultaneous CASP8AP2 gene deletion/insertion, which improved cell viability and protein expression.
ABSTRACT
Synthetic biology aims to contribute to the development of next-generation patient-specific cell-based therapies for chronic diseases especially through the construction of sophisticated synthetic gene switches to enhance the safety and spatiotemporal controllability of engineered cells. Indeed, switches that sense and process specific cues, which may be either externally administered triggers or endogenous disease-associated molecules, have emerged as powerful tools for programming and fine-tuning therapeutic outputs. Living engineered cells, often referred to as designer cells, incorporating such switches are delivered to patients either as encapsulated cell implants or by infusion, as in the case of the clinically approved CAR-T cell therapies. Here, we review recent developments in synthetic gene switches responsive to molecular stimuli, spanning regulatory mechanisms acting at the transcriptional, translational, and posttranslational levels. We also discuss current challenges facing clinical translation of cell-based therapies employing these devices.
ABSTRACT
Chinese hamster ovary (CHO) cells are the most widely used for therapeutic antibody production. In cell line development, engineering secretion processes such as folding-related protein upregulation is an effective way of constructing cell lines with high recombinant protein productivity. However, there have been few studies on the transport of recombinant proteins between the endoplasmic reticulum (ER) and the Golgi apparatus. In this study, Sar1A, a protein involved in COPII vesicle formation, was focused on to improve antibody productivity by enhancing COPII vesicle-mediated antibody transport from the ER to the Golgi apparatus, and to clarify its effect on the secretion process. The constructed Sar1A-overexpressing CHO cell lines were batch-cultured, in which they showed an increased specific antibody production rate. The intracellular antibody accumulation and the specific localization of the intracellular antibodies were investigated by chase assay using a translation inhibitor and observed by immunofluorescence-based imaging analysis. The results showed that Sar1A overexpression reduced intracellular antibody accumulation, especially in the ER. The effects of the engineered antibody transport on the antibody's glycosylation profile and the unfolded protein response (UPR) pathway were analyzed by liquid chromatography-mass spectrometry and UPR-related gene expression evaluation, respectively. Sar1A overexpression lowered glycan galactosylation and induced a stronger UPR at the end of the batch culture. Sar1A overexpression enhanced the antibody productivity of CHO cells by modifying their secretion process. This approach could also contribute to the production of not only monoclonal antibodies but also other therapeutic proteins that require transport by COPII vesicles.
Subject(s)
Cricetulus , Endoplasmic Reticulum , Golgi Apparatus , Recombinant Proteins , CHO Cells , Animals , Endoplasmic Reticulum/metabolism , Recombinant Proteins/metabolism , Recombinant Proteins/genetics , Golgi Apparatus/metabolism , Glycosylation , Cricetinae , Unfolded Protein Response , COP-Coated Vesicles/metabolism , Antibodies, Monoclonal/metabolism , Antibodies, Monoclonal/genetics , Protein Transport , Batch Cell Culture Techniques/methodsABSTRACT
Microglia play vital roles in embryonic and post-natal development, homeostasis, and pathogen defence in the central nervous system. Human induced pluripotent stem cell (hiPSC)-based methods have emerged as an important source for the study of human microglia in vitro. Classical approaches to differentiate hiPSCs into microglia suffer from limitations including extended culture periods, consistency, and efficiency. More recently, forward programming has arisen as a promising alternative for the manufacture of bulk quantities of human microglia. This review provides a comprehensive assessment of published forward programming protocols that are based on forced expression of key lineage transcription factors (TFs). We focus on the choice of reprogramming factors, transgene delivery methods, and medium composition, which impact induction kinetics and the resulting microglia phenotype.
ABSTRACT
Introduction: Wound healing is characterized as a complicated and sophisticated biological process through which tissue heals and repairs itself after injury. However, the normal wound healing process relies on different growth factors as well as the presence of an accurate cytokine level to ensure appropriate cellular responses. In the case of wound healing, the effects of various growth factors have been studied, but the effects of transforming growth factor beta (TGF-ß) on wound healing have been found to be more significant because of its broad spectrum of impacts on healing the wounded tissues or skins. Methods: In the current study, the impact of TGF-ß3 in bone cells' wound healing was examined in vitro. Furthermore, the activities and characteristics of TGF-ß3, as well as those of related growth factors throughout this wound healing process, were studied under hydrodynamic shear stress conditions as well as static conditions of cultured bone cells. Results: We demonstrated that a positive outcome of TGF-ß3 treatment was found after 24 h under a static condition, while TGF-ß3 treatment was found to be effective under a dynamic condition for wound closure. In the case of the dynamic condition, a full wound closure was obtained after 18 h in both the control and TGF-ß3 treatment, while in the case of static conditions, wounds were found to remain open, even after 24 h, for both the control and TGF-ß3 treatment. Additionally, in the static condition, the wound closure rate with TGF-ß3 treatment was found to be quicker than that of the control flask, which implies that wound healing can be postponed in the static condition. In the dynamic condition, the wound healing process became more rapid in a cultured cell environment. Conclusion: The synergistic effect of TGF-ß3 and hydrodynamic shear stress conditions had a positive impact on increasing wound healing and improving the rate of wound closure.
ABSTRACT
BACKGROUND: Adoptive cell therapy, such as chimeric antigen receptor (CAR)-T cell therapy, has improved patient outcomes for hematological malignancies. Currently, four of the six FDA-approved CAR-T cell products use the FMC63-based αCD19 single-chain variable fragment, derived from a murine monoclonal antibody, as the extracellular binding domain. Clinical studies demonstrate that patients develop humoral and cellular immune responses to the non-self CAR components of autologous CAR-T cells or donor-specific antigens of allogeneic CAR-T cells, which is thought to potentially limit CAR-T cell persistence and the success of repeated dosing. METHODS: In this study, we implemented a one-shot approach to prevent rejection of engineered T cells by simultaneously reducing antigen presentation and the surface expression of both Classes of the major histocompatibility complex (MHC) via expression of the viral inhibitors of transporter associated with antigen processing (TAPi) in combination with a transgene coding for shRNA targeting class II MHC transactivator (CIITA). The optimal combination was screened in vitro by flow cytometric analysis and mixed lymphocyte reaction assays and was validated in vivo in mouse models of leukemia and lymphoma. Functionality was assessed in an autologous setting using patient samples and in an allogeneic setting using an allogeneic mouse model. RESULTS: The combination of the Epstein-Barr virus TAPi and an shRNA targeting CIITA was efficient and effective at reducing cell surface MHC classes I and II in αCD19 'stealth' CAR-T cells while retaining in vitro and in vivo antitumor functionality. Mixed lymphocyte reaction assays and IFNγ ELISpot assays performed with T cells from patients previously treated with autologous αCD19 CAR-T cells confirm that CAR T cells expressing the stealth transgenes evade allogeneic and autologous anti-CAR responses, which was further validated in vivo. Importantly, we noted anti-CAR-T cell responses in patients who had received multiple CAR-T cell infusions, and this response was reduced on in vitro restimulation with autologous CARs containing the stealth transgenes. CONCLUSIONS: Together, these data suggest that the proposed stealth transgenes may reduce the immunogenicity of autologous and allogeneic cellular therapeutics. Moreover, patient data indicate that repeated doses of autologous FMC63-based αCD19 CAR-T cells significantly increased the anti-CAR T cell responses in these patients.
Subject(s)
Immunotherapy, Adoptive , Receptors, Chimeric Antigen , Animals , Humans , Mice , Receptors, Chimeric Antigen/immunology , Receptors, Chimeric Antigen/genetics , Immunotherapy, Adoptive/methods , Transgenes , T-Lymphocytes/immunologyABSTRACT
Paclitaxel, a rare diterpene extracted from the bark of Chinese yew (Taxus chinensis), is renowned for its anti-cancer activity and serves as a primary drug for treating cancers. Due to the exceptionally low content of paclitaxel in the bark, a semi-synthetic method that depletes Chinese yew resources is used in the production of paclitaxel, which, however, fails to meet the escalating clinical demand. In recent years, researchers have achieved significant progress in heterologous biosynthesis and metabolic engineering for the production of paclitaxel. This article comprehensively reviews the advancements in paclitaxel production, encompassing chemical synthesis, heterologous biosynthesis, and cell engineering. It provides an in-depth introduction to the biosynthetic pathway and transcriptional regulation mechanisms of paclitaxel, aiming to provide a valuable reference for further research on paclitaxel biosynthesis.
Subject(s)
Paclitaxel , Paclitaxel/biosynthesis , Metabolic Engineering/methods , Taxus/genetics , Taxus/metabolism , Antineoplastic Agents, Phytogenic/biosynthesis , Antineoplastic Agents, Phytogenic/pharmacology , Transcription, Genetic , Biosynthetic Pathways/geneticsABSTRACT
T-cell therapy has emerged as an effective approach for treating viral infections and cancers. However, a significant challenge is the selection of T-cell receptors (TCRs) that exhibit the desired functionality. Conventionally in vitro techniques, such as peptide sensitivity measurements and cytotoxicity assays, provide valuable insights into TCR potency but are labor-intensive. In contrast, measuring ligand binding properties (z-Movi technology) could provide an accelerated processing while showing robust correlations with T-cell functions. In this study, we assessed whether cell avidity can predict functionality also in the context of TCR-engineered T cells. To this end, we developed a flexible system for TCR re-expression by generating a Jurkat-derived T cell clone lacking TCR and CD3 expression through CRISPR-Cas9-mediated TRBC knockout. The knockin of a transgenic TCR into the TRAC locus restored TCR/CD3 expression, allowing for CD3-based purification of TCR-engineered T cells. Subsequently, we characterized these engineered cell lines by functional readouts, and assessment of binding properties through the z-Movi technology. Our findings revealed a strong correlation between the cell avidities and functional sensitivities of Jurkat TCR-T cells. Altogether, by integrating cell avidity measurements with our versatile T cell engineering platform, we established an accelerated system for enhancing the in vitro selection of clinically relevant TCRs.
Subject(s)
Receptors, Antigen, T-Cell , Humans , Receptors, Antigen, T-Cell/metabolism , Receptors, Antigen, T-Cell/immunology , Jurkat Cells , T-Lymphocytes/immunology , T-Lymphocytes/metabolism , T-Lymphocytes/cytology , CRISPR-Cas Systems/genetics , CD3 Complex/metabolism , CD3 Complex/immunologyABSTRACT
The clinical potential of current FDA-approved chimeric antigen receptor (CAR)-engineered T (CAR-T) cell therapy is encumbered by its autologous nature, which presents notable challenges related to manufacturing complexities, heightened costs, and limitations in patient selection. Therefore, there is a growing demand for off-the-shelf universal cell therapies. In this study, we have generated universal CAR-engineered NKT (UCAR-NKT) cells by integrating iNKT TCR engineering and HLA gene editing on hematopoietic stem cells (HSCs), along with an ex vivo, feeder-free HSC differentiation culture. The UCAR-NKT cells are produced with high yield, purity, and robustness, and they display a stable HLA-ablated phenotype that enables resistance to host cell-mediated allorejection. These UCAR-NKT cells exhibit potent antitumor efficacy to blood cancers and solid tumors, both in vitro and in vivo, employing a multifaceted array of tumor-targeting mechanisms. These cells are further capable of altering the tumor microenvironment by selectively depleting immunosuppressive tumor-associated macrophages and myeloid-derived suppressor cells. In addition, UCAR-NKT cells demonstrate a favorable safety profile with low risks of graft-versus-host disease and cytokine release syndrome. Collectively, these preclinical studies underscore the feasibility and significant therapeutic potential of UCAR-NKT cell products and lay a foundation for their translational and clinical development.
Subject(s)
Hematopoietic Stem Cells , Immunotherapy, Adoptive , Natural Killer T-Cells , Receptors, Chimeric Antigen , Humans , Hematopoietic Stem Cells/metabolism , Hematopoietic Stem Cells/cytology , Hematopoietic Stem Cells/immunology , Animals , Receptors, Chimeric Antigen/immunology , Receptors, Chimeric Antigen/genetics , Receptors, Chimeric Antigen/metabolism , Immunotherapy, Adoptive/methods , Mice , Natural Killer T-Cells/immunology , Natural Killer T-Cells/metabolism , Gene Editing , Xenograft Model Antitumor Assays , Neoplasms/therapy , Neoplasms/immunology , Cell Line, Tumor , Receptors, Antigen, T-Cell/metabolism , Receptors, Antigen, T-Cell/genetics , Receptors, Antigen, T-Cell/immunologyABSTRACT
Chimeric antigen receptor T (CAR-T) cell therapy is regarded as a potent immunotherapy and has made significant success in hematologic malignancies by eliciting antigen-specific immune responses. However, response rates of CAR-T cell therapy against solid tumors with immunosuppressive microenvironments remain limited. Co-engineering strategies are advancing methods to overcome immunosuppressive barriers and enhance antitumor responses. Here, we engineered an IL-2 mutein co-engineered CAR-T for the improvement of CAR-T cells against solid tumors and the efficient inhibition of solid tumors. We equipped the CAR-T cells with co-expressing both tumor antigen-targeted CAR and a mutated human interleukin-2 (IL-2m), conferring enhanced CAR-T cells fitness in vitro, reshaped immune-excluded TME, enhanced CAR-T infiltration in solid tumors, and improved tumor control without significant systemic toxicity. Overall, this subject demonstrates the universal CAR-T cells armed strategy for the development and optimization of CAR-T cells against solid tumors.
Subject(s)
Immunotherapy, Adoptive , Interleukin-2 , Neoplasms , Receptors, Chimeric Antigen , T-Lymphocytes , Humans , Interleukin-2/genetics , Interleukin-2/immunology , Receptors, Chimeric Antigen/immunology , Receptors, Chimeric Antigen/genetics , Immunotherapy, Adoptive/methods , Animals , Neoplasms/therapy , Neoplasms/immunology , Neoplasms/genetics , Mice , T-Lymphocytes/immunology , T-Lymphocytes/metabolism , Tumor Microenvironment/immunology , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/immunology , Antigens, Neoplasm/immunology , Antigens, Neoplasm/genetics , Cell Line, Tumor , Xenograft Model Antitumor AssaysABSTRACT
In multiple myeloma (MM), B cell maturation antigen (BCMA)-directed CAR T cells have emerged as a novel therapy with potential for long-term disease control. Anti-BCMA CAR T cells with a CD8-based transmembrane (TM) and CD137 (41BB) as intracellular costimulatory domain are in routine clinical use. As the CAR construct architecture can differentially impact performance and efficacy, the optimal construction of a BCMA-targeting CAR remains to be elucidated. Here, we hypothesized that varying the constituents of the CAR structure known to impact performance could shed light on how to improve established anti-BCMA CAR constructs. CD8TM.41BBIC-based anti-BCMA CAR vectors with either a long linker or a short linker between the light and heavy scFv chain, CD28TM.41BBIC-based and CD28TM.CD28IC-based anti-BCMA CAR vector systems were used in primary human T cells. MM cell lines were used as target cells. The short linker anti-BCMA CAR demonstrated higher cytokine production, whereas in vitro cytotoxicity, T cell differentiation upon activation and proliferation were superior for the CD28TM.CD28IC-based CAR. While CD28TM.CD28IC-based CAR T cells killed MM cells faster, the persistence of 41BBIC-based constructs was superior in vivo. While CD28 and 41BB costimulation come with different in vitro and in vivo advantages, this did not translate into a superior outcome for either tested model. In conclusion, this study showcases the need to study the influence of different CAR architectures based on an identical scFv individually. It indicates that current scFv-based anti-BCMA CAR with clinical utility may already be at their functional optimum regarding the known structural variations of the scFv linker.
Subject(s)
Multiple Myeloma , Humans , Multiple Myeloma/therapy , B-Cell Maturation Antigen , Antibodies , CD28 Antigens , Cell- and Tissue-Based TherapyABSTRACT
BACKGROUND: A bidirectional promoter-driven chimeric antigen receptor (CAR) cassette provides the simultaneous expression of two CARs, which significantly enhances dual antigen-targeted CAR T-cell therapy. METHODS: We developed a second-generation CAR directing CD19 and CD20 antigens, incorporating them in a head-to-head orientation from a bidirectional promoter using a single Sleeping Beauty transposon system. The efficacy of bidirectional promoter-driven dual CD19 and CD20 CAR T cells was determined in vitro against cell lines expressing either, or both, CD19 and CD20 antigens. In vivo antitumor activity was tested in Raji lymphoma-bearing immunodeficient NOD-scid IL2Rgammanull (NSG) mice. RESULTS: Of all tested promoters, the bidirectional EF-1α promoter optimally expressed transcripts from both sense (CD19-CAR) and antisense (GFP.CD20-CAR) directions. Superior cytotoxicity, cytokine production and antigen-specific activation were observed in vitro in the bidirectional EF-1α promoter-driven CD19/CD20 CAR T cells. In contrast, a unidirectional construct driven by the EF-1α promoter, but using self-cleaving peptide-linked CD19 and CD20 CARs, showed inferior expression and in vitro function. Treatment of mice bearing advanced Raji lymphomas with bidirectional EF-1α promoter-driven CD19/CD20 CAR T cells effectively controlled tumor growth and extended the survival of mice compared with group treated with single antigen targeted CAR T cells. CONCLUSION: The use of bidirectional promoters in a single vector offers advantages of size and robust CAR expression with the potential to expand use in other forms of gene therapies like CAR T cells.