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The Emerging Landscape of Immune Cell Therapies
Abstract
Cell therapies present an entirely new paradigm in drug development. Within this class, immune cell therapies are among the most advanced, having already demonstrated definitive evidence of clinical benefits in cancer and infectious disease. Numerous features distinguish these “living therapies” from traditional medicines, including their ability to expand and contract in proportion to need and to mediate therapeutic benefits for months or years following a single application. Continued advances in fundamental immunology, genetic engineering, gene editing, and synthetic biology exponentially expand opportunities to enhance the sophistication of immune cell therapies, increasing potency and safety and broadening their potential for treatment of disease. This perspective will summarize the current status of immune cell therapies for cancer, infectious disease, and autoimmunity, and discuss advances in cellular engineering to overcome barriers to progress.
... Concurrently, significant advances have been made in utilizing immune cells as a novel form of immunotherapy. This progress involves isolating tumor-reactive immune cells from patients, expanding them ex vivo, and then reintroducing them into the patient's body [9]. A key development in this area is the genetic engineering of immune cells, particularly the creation of chimeric antigen receptor T (CAR-T) cells designed to target specific cancer antigens [9,10]. ...
... This progress involves isolating tumor-reactive immune cells from patients, expanding them ex vivo, and then reintroducing them into the patient's body [9]. A key development in this area is the genetic engineering of immune cells, particularly the creation of chimeric antigen receptor T (CAR-T) cells designed to target specific cancer antigens [9,10]. Although CAR-T cells have garnered considerable clinical attention and support, side effects such as cytokine release syndrome and issues related to the tumorigenesis of administered T cells have been reported [10,11]. ...
... Monoclonal antibodies (mAbs) have become a cornerstone of clinical oncolo munotherapy, serving as effective vehicles for the delivery of cytotoxic agents [6] the past twenty years, the development of antibody-drug conjugates (ADCs) ha remarkable, offering promising clinical results by enhancing the delivery of poten cancer drugs while reducing off-target side effects [7,8]. Concurrently, significa vances have been made in utilizing immune cells as a novel form of immunotherap progress involves isolating tumor-reactive immune cells from patients, expanding ex vivo, and then reintroducing them into the patient's body [9]. A key developm this area is the genetic engineering of immune cells, particularly the creation of ch antigen receptor T (CAR-T) cells designed to target specific cancer antigens [9,10 hough CAR-T cells have garnered considerable clinical attention and support, side such as cytokine release syndrome and issues related to the tumorigenesis of admini T cells have been reported [10,11]. ...
While traditional combination anticancer treatments have shown promising results, there remains significant interest in developing innovative methods to enhance and integrate chemotherapy and immunotherapy. This study introduces a recombinant fusion protein-based cell surface modification system that synergistically combines chemotherapy and immunotherapy into a single-targeted chemo-immunotherapy approach. A cell surface-modified protein composed of an antibody-specific binding domain and a cell-penetrating domain rapidly converts immune cells into chemo-immuno therapeutics by binding to antibodies on the surface of immune cells. Utilizing a non-invasive, non-toxic approach free of chemical modifications and binding, our system homogeneously transforms immune cells by transiently introducing targeted cytotoxic drugs into them. The surface-engineered immune cells loaded with antibody–drug conjugates (ADCs) significantly inhibit the growth of target tumors and enhance the targeted elimination of cancer cells. Therefore, NK cells modified by the cell surface-modified protein to incorporate ADCs could be expected to achieve the combined effects of targeted cancer cell recognition, chemotherapy, and immunotherapy, thereby enhancing their therapeutic efficacy against cancer. This strategy allows for the efficient and rapid preparation of advanced chemo-immuno therapeutics to treat various types of cancer and provides significant potential to improve the efficacy of cancer treatment.
... Among these potential risk factors, immune responses and changes in immune cell profiles are associated with the occurrence and progression of VTE [3][4][5][6]. Immune cells such as lymphocytes (B cells and T cells), macrophages/monocytes, and granulocytes are crucial for human defense and participate in various inflammatory reactions [7]. Studies have observed thrombotic immune cell infiltration and changes in systemic circulating immune cell proportions in deep vein thrombosis (DVT) patients [4,8]. ...
... Current research on the relationship between venous thromboembolism (VTE) and immune cells primarily depends on observational cohort analyses and basic experiments [3][4][5][6][7][8][9][10][11][12][13]. Considering that VTE involves multiple risk factors, observational studies can be influenced by confounding factors and reverse causation. ...
Background
Venous thromboembolism (VTE) is a common vascular disease with a significant global burden, influenced by multiple factors, such as genetic, environmental, and immune components. Immune responses and shifts in immune cell profiles are closely linked to the development and progression of VTE, yet current studies are limited by confounding factors and reverse causation. To address these limitations, this study uses Mendelian randomization to explore the causal relationship between immune cell traits and VTE, aiming to provide insights into underlying mechanisms.
Methods
We utilized GWAS data on 731 immunological traits (n = 3757) from the IEU OpenGWAS project and VTE (21021 cases, 391160 controls) from Finngen public data. Five commonly used Mendelian randomization (MR) methods were employed, including inverse-variance weighted (IVW), MR-Egger regression, weighted median estimator (WME), and both weighted and simple models to analyze their associations. Sensitivity checks for the results included pleiotropy tests, heterogeneity tests, and leave-one-out analyses.
Results
From a strictly statistical perspective, no significant associations were observed after FDR correction. However, our exploratory analysis suggested potential trends between immune cell traits and VTE. When immune cells were considered as the exposure and VTE as the outcome, 44 immune cell traits were suggestively associated with VTE based on uncorrected p-values. Conversely, when VTE was considered as the exposure, it appeared to influence immune cell traits. Specifically, secreting CD4 regulatory T cells (OR = 0.9084; 95% CI: 0.8418–0.9804; P = 0.0135; FDR = 0.7339) and activated and resting CD4 regulatory T cells (OR = 0.9275; 95% CI: 0.8622–0.9977; P = 0.0433; FDR = 0.8048) suggested a potential protective trend against VTE. On the other hand, B cells expressing CD20 (OR = 1.0697; 95% CI: 1.0227–1.1188; P = 0.0033; FDR = 0.5767) and myeloid cells expressing CD33 (OR = 1.0199; 95% CI: 1.0021–1.0382; P = 0.0296; FDR = 0.7339) may be linked to an increased risk of VTE.
Conclusions
From a strict statistical perspective, no significant associations were identified after FDR correction. However, our analysis using MR method suggests a potential link between VTE and immune cell traits, suggesting the complex interplay between the immune system and thrombotic events. While this study is exploratory and needs validation, the findings of this study are hypothesis-generating with resect to the mechanisms underlying VTE and encourage further investigation into the role of immune activity in VTE pathology.
Graphical abstract
... This mixture allows for large-scale expansion of the original lymphocyte population [67,69]. Finally, the solution is administered back to the patient with systemic IL-2 or in combination with other chemotherapeutics (Figure 3) [71,72]. ...
... This mixture allows for large-scale expansion of the original lymphocyte population [67,69]. Finally, the solution is administered back to the patient with systemic IL-2 or in combination with other chemotherapeutics (Figure 3) [71,72]. The idea that cell-mediated immunity could play a role in cancer therapy began with studies looking at IL-2, a cytokine that modulates the immune response. ...
Immunotherapy offers a novel and promising option in the treatment of late-stage melanoma. By utilizing the immune system to assist in tumor destruction, patients have additional options after tumor progression. Immune checkpoint inhibitors reduce the ability for tumors to evade the immune system by inhibiting key surface proteins used to inactivate T-cells. Without these surface proteins, T-cells can induce cytotoxic responses against tumors. Tumor infiltrating lymphocyte therapy is a form of adoptive cell therapy that takes advantage of a small subset of T-cells that recognize and infiltrate tumors. Isolation and rapid expansion of these colonies assist the immune system in mounting a charged response that can induce remission. Tumor vaccines deliver a high dose of unique antigens expressed by tumor cells to the entire body. The introduction of large quantities of tumor antigens upregulates antigen presenting cells and leads to effective activation of the immune system against tumors. Cytokine therapy introduces high amounts of chemical messengers that are endogenous to the immune system and support T-cell expansion. While other methods of immunotherapy exist, immune checkpoint inhibitors, tumor infiltrating lymphocytes, tumor vaccines, and cytokine therapy are commonly used to treat melanoma. Like many other cancer treatments, immunotherapy is not without adverse effects, as toxicities represent a major obstacle. However, immunotherapy has been efficacious in the treatment of melanoma.
... With the continuous advancements in immunobiology and synthetic biology, along with the rapid progress of clinical-scale genetic engineering and gene editing technologies, various emerging cellular therapies have significantly advanced tumor therapy and other diseases (11). Cellular therapy has gradually become an important branch in the field of tumor treatment. ...
... HSCs, as multipotent stem cells capable of differentiating into various immune cells, offer a new perspective on immune modulation. CAR-T cells have shown promise in the treatment of autoimmune diseases by eliminating pathologically activated immune cells or re-establishing immune tolerance in affected organs (11). CAAR-T cells and CAR-NK cells utilize their specificity and enhanced targeting capabilities to provide innovative avenues for the treatment of autoimmune diseases. ...
Autoimmune diseases occur due to a dysregulation within the immune system, leading to an aberrant assault on the organism’s own tissues. The pathogenesis of these conditions is multifactorial, encompassing intricate interplays among genetic predispositions, environmental determinants, and hormonal fluctuations. The spectrum of autoimmune diseases is broad, impacting a multitude of organ systems, with notable examples such as rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), multiple sclerosis (MS), psoriasis, and vitiligo. Despite substantial progress in therapeutic interventions over recent years, a definitive cure for autoimmune diseases has yet to be realized, with existing modalities largely providing palliative care. Cellular therapy is considered the fourth pillar in the management of oncological disorders subsequent to surgical resection, radiotherapy, and chemotherapy. Cellular therapies have shown potential in augmenting immune competence and eliminating of targeted neoplastic cells in a spectrum of cancers. As targeting specific molecules on the surface of autoreactive B and T cells, such as CD19, BCMA, CD20, and CTLA-4, cellular therapies are emerging as promising approaches for the treatment of autoimmune diseases. This review delineates the advancements in the application of cellular therapies applied recently for autoimmune diseases and proposes considerations for the advancement of novel therapeutic strategies.
... Innovative T-cell-based immunotherapies have demonstrated significant potential in tumor immunotherapy. Chimeric antigen receptor (CAR) T cells, engineered through genetic modification to acquire tumor-specific recognition and cytotoxic capabilities, represent a prominent example of this approach 9 . The CAR structure is an artificially designed antigen-recognition receptor, with thirdgeneration CAR constructs currently being the most clinically utilized configuration. ...
Glioblastoma (GBM) represents the most prevalent and aggressive primary malignant neoplasm in the adult central nervous system, exhibiting marked infiltrative growth patterns, inevitable recurrence, and dismal therapeutic outcomes with current treatment modalities. While CAR-T cell immunotherapy has demonstrated remarkable success in hematological malignancies, its clinical translation for GBM has been hampered by several fundamental limitations. A key factor among these is tumor-intrinsic heterogeneity, which drives antigen escape and therapeutic resistance. Furthermore, although autologous CAR-T approaches dominate current clinical investigations, they encounter substantial barriers including manufacturing variability, scalability constraints, and practical limitations for widespread clinical deployment. In contrast, allogeneic "off-the-shelf" CAR-T therapy holds greater potential for the future applications. γδ T cells are a particularly compelling candidate for universal CAR therapy, offering several advantages including innate MHC-unrestricted target recognition obviating the need for HLA matching, polyfunctional cytotoxic mechanisms capable of addressing heterogeneous tumor populations, and intrinsic tropism for solid tumors. However, translational implementation has been constrained by their physiological rarity, ex vivo expansion difficulties, and genetic modification inefficiencies. To address these challenges, we adopted a dual-pronged targeting strategy focusing on B7-H3 and IL13Rα2 - two surface antigens demonstrating preferential overexpression across GBM subtypes while maintaining limited distribution in normal tissues. Using phage display platform and function-based nanobody screening, we identified high-affinity binders against both targets. Subsequent optimization of γδ T cell expansion protocols and lentiviral transduction parameters enabled the development of a bispecific, allogeneic CAR-γδ T cell platform. Our in vitro studies revealed that dual-target CAR-γδT cells sustained proliferative capacity under GMP-compatible culture conditions, exhibited potent and specific cytotoxicity against antigen-positive glioma cells, and critically, they showed superior elimination of target-heterogeneous tumors compared to monospecific CAR-T constructs. These results establish a robust preclinical foundation for clinical translation and highlight the therapeutic potential of combinatorial antigen targeting coupled with allogeneic γδ T cell engineering to overcome the persistent challenges in GBM immunotherapy.
... Gene therapy, an emerging immunotherapy method, aims to repair or replace disease-causing genes. It works through three main mechanisms: (1) Replacement: Replacing the faulty gene with a normal one to achieve stable, controllable, and targeted expression, thereby protecting and repairing damaged tissues; (2) Silencing: Silencing the expression of malfunctioning genes; (3) Insertion: Introducing a new or modified gene [148]. Gene therapy can use gene delivery, gene editing, and gene regulation methods to reduce local inflammation around implants, inhibit bone resorption, and promote new bone formation, thereby alleviating PI symptoms and offering a novel treatment approach [149,150]. ...
As dental implant technology becomes increasingly widespread, peri-implantitis induced bone resorption and implant loss have garnered significant academic attention. Current clinical treatments for peri-implantitis primarily focus on plaque control, but the limitations of traditional therapies often hinder effective outcomes. Treatment strategies targeting immune responses that can eliminate inflammation, control osteolytic environments, and restore physiological bone formation are promising approaches. This article comprehensively reviews the role of the immune system in the pathogenesis and progression of peri-implantitis through a synthesis of multiple literature sources. It introduces current immunomodulatory strategies in the treatment of peri-implantitis and discusses the potential applications and challenges of novel immunotherapies, including gene therapy, cell engineering, and nanotechnology, in the management of peri-implantitis. The aim is to provide guidance for translating immunotherapies from the laboratory to clinical practice.
... CAR-T therapy can potentially be used for the treatment of cancer, infectious diseases, autoimmune diseases, cardiac diseases, and others [8,9]. Furthermore, CAR technology has recently been used to develop novel ACTs with other types of immune cells, including natural killer cells and their subtypes, macrophages, or gamma delta T cells [10][11][12]. ...
Chimeric antigen receptor T-cell (CAR-T) therapies are one of the main approaches among targeted cellular therapies. Despite the potential benefit and durable responses observed in some patients receiving CAR-T therapies, serious and potentially fatal toxicities remain a major challenge. The most common CAR-T-associated toxicities include cytokine release syndrome (CRS), neurotoxicity, cytopenias, and infections. While CRS and neurotoxicity are generally managed with tocilizumab and corticosteroids, respectively, high-grade toxicities can be life-threatening. Close postinfusion monitoring and assessment of clinical laboratory parameters, patient-related and clinical risk factors (e.g., age, tumor burden, comorbidities, baseline laboratory parameters, and underlying abnormalities), and therapy-related risk factors (e.g., CAR-T type, dose, and CAR-T-induced toxicity) are effective strategies to mitigate the toxicities. Clinical laboratory parameters, including various cytokines, have been identified for CRS (interleukin [IL]-1, IL-2, IL-5, IL-6, IL-8, IL-10, C-reactive protein [CRP], interferon [IFN]-γ, ferritin, granulocyte-macrophage colony-stimulating factor [GM-CSF], and monocyte chemoattractant protein-1), neurotoxicity (IL-1, IL-2, IL-6, IL-15, tumor necrosis factor [TNF]-α, GM-CSF, and IFN-γ), cytopenias (IL-2, IL-4, IL-6, IL-10, IFN-γ, ferritin, and CRP), and infections (IL-8, IL-1β, CRP, IFN-γ, and procalcitonin). CAR-T-associated toxicities can be monitored and treated to mitigate the risk to patients. Assessment of alterations in clinical laboratory parameter values that are correlated with CAR-T-associated toxicities may predict development and/or severity of a given toxicity, which can improve patient management strategies and ultimately enable the patients to better tolerate these therapies.
... Unlike TIL therapy, TCR-T therapy relies on the isolation and genetic modification of peripheral blood T cells via apheresis, followed by ex vivo transduction with a tumor-specific receptor targeting melanoma-associated antigens [68]. TCR-T has elicited intense investigation interest since CAR-T therapy failed to meet the need for most solid tumors [69]. Therefore, TCR-T is considered another promising ACT modality for advanced melanoma, due to its highly immunogenetic next to the TIL therapy. ...
Cutaneous melanoma is one of the most aggressive skin cancers originating from skin pigment cells. Patients with advanced melanoma suffer a poor prognosis and generally cannot benefit well from surgical resection and chemo/target therapy due to metastasis and drug resistance. Thus, adoptive cell therapy (ACT), employing immune cells with specific tumor-recognizing receptors, has emerged as a promising therapeutic approach to display on-tumor toxicity. This review discusses the application, efficacy, limitations, as well as future prospects of four commonly utilized approaches -including tumor-infiltrating lymphocytes, chimeric antigen receptor (CAR) T cell, engineered T-cell receptor T cells, and chimeric antigen receptor NK cells- in the context of malignant melanoma.
... Advances in synthetic biology have expanded the biologist horizon to explore the possibilities to engineer and manipulate organisms thereby allowing creating bacteria stains that are genetically altered to have specific therapeutic and biotechnological benefits. 1 These, in turn, have tremendous potential with a new therapeutic power and precision. 2 ...
Synthetic biology is an emerging discipline of science, at the intersection of biology, engineering, and chemistry that involves redesigning organisms to have new phenotypes and customized abilities. While synthetic biology seems to have originated from genetic engineering, over the years, it has matured as well as diverged from it. It involves not just the transfer of genes from one or cell to another creating some variants, it also involves the assembly of an altogether novel organism or cell created part by part by the assembly of individual components of the desired function in a logical fashion. In this minireview, we will explore this new discipline and its possible applications and future promises to serve the humanity
... CAR T cell therapy has revolutionized the treatment of "liquid" hematologic malignancies such as non-Hodgkin lymphomas and multiple myeloma, with six current U.S. Bryan D. Choi bchoi@mgh.harvard.edu FDA-approved CAR T cell products that target two tumor surface antigens, CD19 or BCMA [4]. However, the advent of CAR T cells to treat other cancers has been hampered by the many immunologic challenges that "solid" tumors harness to evade immunity, including anatomic sequestration, a hostile tumor microenvironment (TME), and antigen downregulation or escape [5]. ...
Context
Chimeric antigen receptor (CAR) T cell therapy is an exciting modality of immunotherapy that has revolutionized the treatment of hematologic malignancies. However, translating this success to malignant gliomas such as glioblastoma (GBM) and diffuse midline glioma (DMG) remains a formidable challenge due to multiple biologic, anatomic, and immunologic factors. Despite these hurdles, a number of clinical trials deployed over the last decade have increased optimism for the potential of CAR T cell therapy in glioma treatment.
Evidence synthesis
We highlight historical and ongoing clinical trials of CAR T cell therapy in glioma, with a focus on key tumor-associated antigens such as IL-13Rα2, HER2, EGFR, EGFRvIII, EphA2, GD2, and B7-H3. Early studies established proof-of-concept for antigen-specific CAR T cell targeting, yet immune evasion mechanisms such as antigen downregulation and limited CAR T cell persistence remain significant obstacles. Recent approaches, including multiantigen targeting, alternative cell sources, and innovations in delivery routes offer promising strategies to overcome these challenges.
Conclusions
The rapid evolution of investigational CAR T cell therapies portends great potential for the future of glioma treatment. Future studies will need to refine antigen targeting strategies, optimize CAR T cell persistence, and integrate combinatorial approaches to fully harness the therapeutic potential of this modality and improve the therapeutic window against brain tumors.
... By doing so, we can create a future where genetic medicine reigns supreme and diseases become mere fragments of the past. With diligent efforts and unwavering determination, we shall forge a path towards a brighter tomorrow, where the advancements in genetic therapies shape the very essence of our existence and hold within them the power to heal, transform, and nurture the entirety of the human race [115,116,41,117,118,119] . ...
... The recent introduction of advanced therapeutic medicinal products to cancer treatment and regenerative medicine has had a substantial contribution to patient health, but faces significant challenges in manufacture as they are based on the use of living cells rather than drugs or biomolecules. These cell-based treatments generally require complex manufacturing procedures to go from source material through to their final formulation (Weber et al. 2020). Regenerative cell therapies involve one or more stages including the isolation, expansion, manipulation and Tom Carvell trc2000@hw.ac.uk 1 importance of buffer exchange within the manufacturing process due to the numerous times that changing the fluid environment of cells is required. ...
Buffer exchange is a common process in manufacturing protocols for a wide range of bioprocessing applications, with a variety of technologies available to manipulate biological materials for culture medium exchange, cell washing and buffer removal. Microfluidics is an emerging field for buffer exchange and has shown promising results with both prototype research and commercialised devices which are inexpensive, highly customisable and often have the capacity for scalability to substantially increase throughput. Microfluidic devices are capable of processing biological materials and exchanging solutions without the need for conventional processing techniques like centrifugation, which are time-consuming, unsuitable for large volumes and may be damaging to cells. The use of microfluidic separation devices for cell therapy manufacturing has been under-explored despite some device designs successfully being used for diagnostic enrichment of rare circulating tumour cells from peripheral blood. This mini-review aims to review the current state of microfluidic devices for buffer exchange, provide an insight into the advantages microfluidics offers for buffer exchange and identify future developments key to exploiting the technology for this application.
... " The prototype of designer immune cell therapy is CAR T-cell therapy. CD19 CAR T-cell therapy targeting CD19 or BCMA has demonstrated overwhelming therapeutic efficacy in patients with B-cell leukemia/lymphoma or multiple myeloma [1,2]. The advent of CAR-T is a historic turning point that has demonstrated that "cell" modalities can be established as a revolutionary treatment modality. ...
... On the contrary, FoxO1 inhibition might limit the efficacy of chimeric antigen receptor (CAR) T cell therapy. CAR T cells' exhaustion and differentiation are major factors reducing their cytotoxicity and anti-tumor effects [152,153]. FoxO1 has been recently described as a key cell factor maintaining metabolic fitness and a 'stemness-like' phenotype that both significantly enhance CAR T cells' cytotoxic capacity/durability. FoxO1 activity also highly correlates with clinical responses to CAR T cells in CLL and ALL [154,155]. ...
FoxO transcription factors (FoxO1, FoxO3a, FoxO4, FoxO6) are a highly evolutionary conserved subfamily of the ‘forkhead’ box proteins. They have traditionally been considered tumor suppressors, but FoxO1 also exhibits oncogenic properties. The complex nature of FoxO1 is illustrated by its various roles in B cell development and differentiation, immunoglobulin gene rearrangement and cell-surface B cell receptor (BCR) structure, DNA damage control, cell cycle regulation, and germinal center reaction. FoxO1 is tightly regulated at a transcriptional (STAT3, HEB, EBF, FoxOs) and post-transcriptional level (Akt, AMPK, CDK2, GSK3, IKKs, JNK, MAPK/Erk, SGK1, miRNA). In B cell malignancies, recurrent FoxO1 activating mutations (S22/T24) and aberrant nuclear export and activity have been described, underscoring the potential of its therapeutic inhibition. Here, we review FoxO1's roles across B cell and myeloid malignancies, namely acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), follicular lymphoma (FL), diffuse large B cell lymphoma (DLBCL), mantle cell lymphoma (MCL), Burkitt lymphoma (BL), Hodgkin lymphoma (HL), and multiple myeloma (MM). We also discuss preclinical evidence for FoxO1 targeting by currently available inhibitors (AS1708727, AS1842856, cpd10).
... Autologous chimeric antigen receptor (CAR) T cell therapy has demonstrated remarkable efficacy in patients with B cell leukemia/ lymphoma and multiple myeloma. [1][2][3][4] However, CAR T cell therapy is extremely expensive. In addition, several weeks are needed to produce CAR T cells from autologous T cells. ...
Cord blood (CB)-derived chimeric antigen receptor (CAR)-natural killer (NK) cells targeting CD19 have been shown to be effective against B cell malignancies. While human CD56⁺ NK cells can be expanded in vitro, NK cells can also be differentiated from hematopoietic progenitor cells. It is still unclear whether CAR-NK cells originate from mature NK cells or NK progenitor cells in CB. Here, we determined that CAR-NK cells were predominantly derived from CD56⁻ NK progenitor cells. We first found that substantial numbers of CD19 CAR-NK cells were produced from CD56⁻ CB mononuclear cells after in vitro culture for 2 weeks. Single-cell RNA sequencing analysis of CD56⁻CD3⁻CD14⁻CD19⁻ CB mononuclear cells revealed that these cells could be subdivided into three subpopulations based on the expression of CD34 and human leukocyte antigen (HLA)-DR. NK cells originated primarily from CD34⁻HLA-DR⁻ cells. In addition, among the CD34⁻HLA-DR⁻ cells, only CD7⁺ cells could differentiate into NK cells. These results indicate that CD56⁻CD7⁺CD34⁻HLA-DR⁻ lineage marker (Lin)⁻ cells are the major origin of human CB-derived CAR-NK cells, indicating the importance of developing methods to enhance the quality and quantity of NK cells produced from these NK progenitor cells.
... Due to spontaneous aggregation of the CAR receptors at the cell surface, some CARs induce tonic signals in the absence of ligand stimulation. [67][68][69][70] Prolonged and high levels of stimulation can lead to CAR-Treg exhaustion, epigenetic changes, and complete loss of suppression in vivo. 70,71 Therefore, the selection of the CAR with the lowest tonic signaling was the main criterion for the development of our IL23R-CAR. ...
Background and Aims
Regulatory T cells (Tregs) are key regulators in maintaining tissue homeostasis. Disrupted immune homeostasis is associated with Crohn’s disease (CD) pathogenesis. Thus, Treg therapy represents a promising long-acting treatment to restore immune balance in the diseased intestine. Chimeric antigen receptor (CAR) T-cell therapy has revolutionized cancer treatment. This innovative approach also provides the opportunity to improve therapy for CD. By targeting a disease-relevant protein, interleukin-23 receptor (IL23R), we engineered Tregs expressing IL23R-CAR for treating active CD.
Methods
Intestinal IL23R expression from active CD was verified by immunohistochemical analysis. Phenotypic and functional characteristics of IL23R-CAR Tregs were assessed using in vitro assays and their migration capacity was monitored in a xenograft tumor model. Transcriptomic and proteomic analyses were performed to associate molecular profiles with IL23R-CAR Treg activation against colon biopsy-derived cells from active CD patients.
Results
Our study showed that IL23R-CAR displayed negligible tonic signaling and a strong signal-to-noise ratio. IL23R-CAR Tregs maintained regulatory phenotype during in vitro expansion, even when chronically exposed to proinflammatory cytokines and target antigen. IL23R engagement on IL23R-CAR Tregs triggered CAR-specific activation and significantly enhanced their suppressive activity. Also, IL23R-CAR Tregs migrated to IL23R-expressing tissue in humanized mice. Finally, IL23R-CAR Tregs elicited a specific activation against colon biopsy-derived cells from active CD, suggesting an efficient CAR engagement in active CD. Molecular profiling of CD patient biopsies also revealed transcriptomic and proteomic patterns associated with IL23R-CAR activation.
Conclusions
Overall, our results demonstrate that IL23R-CAR Tregs represent a promising therapy for active CD.
Advanced biotherapeutic systems such as gene therapy, mRNA lipid nanoparticles, antibody–drug conjugates, fusion proteins, and cell therapy have proven to be promising platforms for delivering targeted biologic therapeutics. Preserving the intrinsic stability of these advanced therapeutics is essential to maintain their innate structure, functionality, and shelf life. Nevertheless, various challenges and obstacles arise during formulation development and throughout the storage period due to their complex nature and sensitivity to various stress factors. Key stability concerns include physical degradation and chemical instability due to various factors such as fluctuations in pH and temperature, which results in conformational and colloidal instabilities of the biologics, adversely affecting their quality and therapeutic efficacy. This review emphasizes key stability issues associated with these advanced biotherapeutic systems and approaches to identify and overcome them. In gene therapy, the brittleness of viral vectors and gene encapsulation limits their stability, requiring the use of stabilizers, excipients, and lyophilization. Keeping cells viable throughout the whole cell therapy process, from culture to final formulation, is still a major difficulty. In mRNA therapeutics, stabilization strategies such as the optimization of mRNA nucleotides and lipid compositions are used to address the instability of both the mRNA and lipid nanoparticles. Monoclonal antibodies are colloidally and conformationally unstable. Hence, buffers and stabilizers are useful to maintain stability. Although fusion proteins and monoclonal antibodies share structural similarities, they show a similar pattern of instability. Antibody–drug conjugates possess issues with conjugation and linker stability. This review outlines the stability issues associated with advanced biotherapeutics and provides insights into the approaches to address these challenges.
CRISPR-Cas9 systems have revolutionized genome editing, but the off-target effects of Cas9 limit its use in clinical applications. Here, we systematically evaluate FrCas9, a variant from Faecalibaculum rodentium , for cell and gene therapy (CGT) applications and compare its performance to SpCas9 and OpenCRISPR-1. OpenCRISPR-1 is a CRISPR system synthesized de novo using large language models (LLMs) but has not yet undergone systematic characterization. Using AID-seq, Amplicon sequencing, and GUIDE-seq, we assessed the on-target activity and off-target profiles of these systems across multiple genomic loci. FrCas9 demonstrated higher on-target efficiency and substantially fewer off-target effects than SpCas9 and OpenCRISPR-1. Furthermore, TREX2 fusion with FrCas9 reduced large deletions and translocations, enhancing genomic stability. Through screening of 1903 sgRNAs targeting 21 CGT-relevant genes using sequential AID-seq, Amplicon sequencing, and GUIDE-seq analysis, we identified optimal sgRNAs for each gene. Our high-throughput screening platform highlights FrCas9, particularly in its TREX2-fused form, as a highly specific and efficient tool for precise therapeutic genome editing.
Acute myeloid leukemia (AML)-specific target antigens are difficult to identify. Here we demonstrate that HLA-DRB1 can serve as a leukemia-specific target of chimeric antigen receptor (CAR) T cells in patients with AML after allogeneic hematopoietic stem cell transplantation (allo-HCT). We identified KG2032 as a monoclonal antibody specifically bound to AML cells in about half of patients, but not to normal leukocytes other than B lymphocytes. KG2032 reacted with a subset of HLA-DRB1 molecules, specifically those in which the 86th amino acid was not aspartic acid. KG2032 reacted minimally with nonhematopoietic tissues. These results indicate that KG2032 reactivity is highly specific for AML cells in patients who carry KG2032-reactive HLA-DRB1 alleles and who received allo-HCT from a donor carrying KG2032-nonreactive HLA-DRB1 alleles. KG2032-derived CAR T or natural killer cells showed significant anti-leukemic activity in preclinical models in female mice, suggesting that they may cure patients with AML who are incurable with allo-HCT.
Cancer immunotherapy, which leverages the body's immune system to combat cancer, offers the promise of lower toxicity and higher therapeutic efficacy compared to conventional treatments. However, current immunotherapeutic approaches face significant challenges including variable patient response, immune‐related adverse events, and high costs, underscoring the urgent need for innovative strategies. Metal‐based nanomaterials have emerged as a promising avenue in cancer immunotherapy due to their unique physicochemical properties and immune‐regulating capabilities. Despite their potential, concerns about toxicity, incomplete understanding of their immune modulation mechanisms, and early‐stage design strategies hinder their clinical translation. This review summarizes recent advancements in metal‐based nanomaterials for cancer immunotherapy, elucidates the mechanisms by which they enhance antitumor immunity responses, and explores the potential synergistic effects of combining multiple metals. We also discuss key challenges and future perspectives for clinical application, aiming to provide a theoretical foundation for the development of metal‐based immunotherapies and to promote their broader application in cancer treatment.
T cell-based immunotherapies hold promise in treating cancer by leveraging the immune system’s recognition of cancer-specific antigens¹. However, their efficacy is limited in tumours with few somatic mutations and substantial intratumoural heterogeneity2, 3–4. Here we introduce a previously uncharacterized class of tumour-wide public neoantigens originating from RNA splicing aberrations in diverse cancer types. We identified T cell receptor clones capable of recognizing and targeting neoantigens derived from aberrant splicing in GNAS and RPL22. In cases with multi-site biopsies, we detected the tumour-wide expression of the GNAS neojunction in glioma, mesothelioma, prostate cancer and liver cancer. These neoantigens are endogenously generated and presented by tumour cells under physiologic conditions and are sufficient to trigger cancer cell eradication by neoantigen-specific CD8⁺ T cells. Moreover, our study highlights a role for dysregulated splicing factor expression in specific cancer types, leading to recurrent patterns of neojunction upregulation. These findings establish a molecular basis for T cell-based immunotherapies addressing the challenges of intratumoural heterogeneity.
In a phase 1 clinical trial open to accrual from 2004 to 2009, we treated children with neuroblastoma with Epstein–Barr virus (EBV)-specific T lymphocytes and CD3-activated T cells—each expressing chimeric antigen receptors (CARs) targeting GD2 but without an embedded co-stimulatory sequence (first-generation CARs). These CARs incorporated barcoded sequences to track each infused population. We previously reported outcomes up to 5 years and now report long-term outcomes up to 18 years. Of 11 patients with active disease at infusion, three achieved a complete response that was sustained in two patients, one for 8 years until lost to follow-up and one for more than 18 years. Of eight patients with no evidence of disease at the time of CAR-T administration, five are disease free at their last follow-up between 10 years and 15 years after infusion. Intermittent low levels of transgene were detected during the follow-up period with significantly greater persistence in those who were long-term survivors. Despite using first-generation vectors that are no longer employed because of the lack of co-stimulatory domains, patients with relapsed/refractory neuroblastoma achieved long-term disease control after receiving GD2 CAR-T cell therapy, including one patient now in remission of relapsed disease for more than 18 years.
ClinicalTrials.gov identifier: NCT00085930.
Hepatocellular carcinoma (HCC) remains one of the most challenging malignancies globally, characterized by significant heterogeneity, late‐stage diagnosis, and resistance to treatment. In recent years, the advent of immune‐checkpoint blockades (ICBs) and targeted immune cell therapies has marked a substantial advancement in HCC treatment. However, the clinical efficacy of these existing therapies is still limited, highlighting the urgent need for new breakthroughs. Natural killer (NK) cells, a subset of the innate lymphoid cell family, have shown unique advantages in the anti‐tumour response. With increasing evidence suggesting the crucial role of dysfunctional NK cells in the pathogenesis and progression of HCC, considerable efforts have been directed toward exploring NK cells as a potential therapeutic target for HCC. In this review, we will provide an overview of the role of NK cells in normal liver immunity and in HCC, followed by a detailed discussion of various NK cell‐based immunotherapies and their potential applications in HCC treatment.
Background:
Previous studies have shown that an increased number of immune cells is closely associated with the onset and course changes of intracerebral hemorrhage, but the exact causal relationship has not been clarified. The aim of this study was to investigate the causal relationship between immune cells and intracerebral hemorrhage by a two-way Mendelian randomization method.
Methods:
Two sets of SNPs were used as instrumental variables and two-way Mendelian randomization analyses were performed and leave-one-out method were used to assess the validity and heterogeneity of the included genetic variation instruments. The level of multiplicity and heterogeneity of the included genetic variance instruments was assessed.
Results:
The results showed a clear causal relationship between three immune cells and intracerebral hemorrhage, and no heterogeneity between SNPs related to intracerebral hemorrhage, while scatterplot and funnel plot confirmed that the causality was less likely to be biased; MR-Egger results suggested that no genetic pleiotropy was found. Leave-one-out analysis was applied to suggest that the MR analysis results for a single SNP were robust; meanwhile, Meta-analysis was applied to combine the two intracerebral hemorrhage datasets, and the analysis results suggested that in the fixed-effects model and random-effects model, the immunocyte CD66b on Granulocytic Myeloid-Derived Suppressor Cells and other three immune cells were significantly causally associated with intracerebral hemorrhage, while the heterogeneity test suggested that there was no significant difference between the different datasets.
Conclusions:
The present study found a significant causal relationship between specific immune cell phenotypes and intracerebral hemorrhage by Mendelian randomization analysis.
Immunotherapy is now an entrenched form of treatment for cancer patients, among the various established one’s such as immune checkpoint inhibitors, monoclonal antibodies, or chimeric antigen receptor (CAR) T-cell therapy. Established agents have shown promising clinical profile for treating various tumor types, but also demonstrated toxicity profiles. New strategies for trial design and selection of drug dose are in demand owing to classic hypothesis foist by cytotoxic agents. The main target of this chapter is compilation of FDA-approved drugs and current stage of study design of ongoing clinical trials of cancer immunotherapeutic agents. For the advancement in the effectuality of cancer immunotherapy, new approaches are needed, which collectively sum up optimal dose for study in later phases, or checking combination of drugs to enhance effectiveness and decrease toxicity.
Secretory autophagy is a classical form of unconventional secretion that integrates autophagy with the secretory process, relying on highly conserved autophagy-related molecules and playing a critical role in tumor progression and treatment resistance. Traditional autophagy is responsible for degrading intracellular substances by fusing autophagosomes with lysosomes. However, secretory autophagy uses autophagy signaling to mediate the secretion of specific substances and regulate the tumor microenvironment (TME). Cytoplasmic substances are preferentially secreted rather than directed toward lysosomal degradation, involving various selective mechanisms. Moreover, substances released by secretory autophagy convey biological signals to the TME, inducing immune dysregulation and contributing to drug resistance. Therefore, elucidating the mechanisms underlying secretory autophagy is essential for improving clinical treatments. This review systematically summarizes current knowledge of secretory autophagy, from initiation to secretion, considering inter-tumor heterogeneity, explores its role across different tumor types. Furthermore, it proposes future research directions and highlights unresolved clinical challenges.
The applications of single-cell and spatial technologies in recent times have revolutionized the present understanding of cellular states and the cellular heterogeneity inherent in complex biological systems. These advancements offer unprecedented resolution in the examination of the functional genomics of individual cells and their spatial context within tissues. In this review, we have comprehensively discussed the historical development and recent progress in the field of single-cell and spatial genomics. We have reviewed the breakthroughs in single-cell multi-omics technologies, spatial genomics methods, and the computational strategies employed toward the analyses of single-cell atlas data. Furthermore, we have highlighted the advances made in constructing cellular atlases and their clinical applications, particularly in the context of disease. Finally, we have discussed the emerging trends, challenges, and opportunities in this rapidly evolving field.
Cell therapy is the transfer of cells into a patient that has emerged as a promising approach to improve or even cure a wide range of diseases. This strategy takes advantage of the use of living cells that interact actively with the host organism to overcome biological barriers to target specific cell types and tissues. Cell therapy could be based on the use of either stem cells, unspecialized and self-renewing cells that can differentiate into several cell types, or non-stem cells/somatic cells highly specialized that can be further manipulated or treated before reintroduction into humans. This chapter outlines the general concept and types of cell therapies, with a focus on two of the most advanced types used in clinical practice: mesenchymal stem cells and chimeric antigen receptor (CAR)-T cells.
This chapter delves into the innovative domain of cell-guided drugs in tumor therapy, showcasing groundbreaking developments in this area. The focus of cell-guided therapy is primarily on two cell types: stem cells and immune cells. While stem cell transplants are a mainstay in treating hematological malignancies, their broader use as drug agents in cancer therapy remains limited. Therefore, this chapter places a special emphasis on adoptive cell therapy (ACT), a more promising approach that leverages immune cells to target and destroy cancer cells. We offer a thorough introduction to ACT, discussing its principal methods, such as Lymphokine-Activated Killer (LAK) cells, Cytokine-Induced Killer (CIK) cells, Tumor-Infiltrating Lymphocytes (TILs), T Cell Receptor (TCR) therapy, Chimeric Antigen Receptor (CAR) T cell therapy, and Natural Killer (NK) cell therapy. Beyond outlining these advanced techniques, the chapter traces their historical development and evolution. It provides a critical analysis of clinical trials, sheds light on the drugs that have gained approval, and explores the future prospects and potential of ACT in the ongoing fight against cancer.
Immunoreceptors have crucial roles in sensing environmental signals and initiating immune responses to protect the host. Dysregulation of immunoreceptor signalling can therefore lead to a range of diseases, making immunoreceptor-based therapies a promising frontier in biomedicine. A common feature of various immunoreceptors is the basic-residue-rich sequence (BRS), which is a largely unexplored aspect of immunoreceptor signalling. The BRS is typically located in the cytoplasmic juxtamembrane region of immunoreceptors, where it forms dynamic interactions with neighbouring charged molecules to regulate signalling. Loss or gain of the basic residues in an immunoreceptor BRS has been linked to severe human diseases, such as immunodeficiency and autoimmunity. In this Perspective, we describe the role of BRSs in various immunoreceptors, elucidating their signalling mechanisms and biological functions. Furthermore, we highlight pathogenic mutations in immunoreceptor BRSs and discuss the potential of leveraging BRS signalling in engineered T cell-based therapies.
Comprehensive Summary
Due to the immunosuppressive tumor microenvironment (TME), T cells are usually inactivated and tend to differentiate into regulatory T cells (Tregs) in the tumor tissues, which seriously hinders the anti‐tumor efficiency of immunotherapy. In this study, an immune‐stimulating polypeptide hydrogel conjugated with an indoleamine‐2,3‐dioxygenase (IDO) inhibitor, 1‐methyl‐ D ‐tryptophan (D1MT), through a glutathione (GSH)‐responsive spacer was developed as an immunotherapy platform capable of regulating the TME. A combined immunotherapy system was further constructed through encapsulating doxorubicin (Dox) and immune checkpoint blocking antibody targeting programmed cell death protein 1 (aPD‐1) in the hydrogel. Dox released from the hydrogel could cause the immunogenic cell death (ICD) of tumor cells and induce the maturation of antigen presenting cells (APCs). After intratumoral injection, the multiple agent‐loaded hydrogel elicited effective anti‐tumor immunity in mice bearing B16F10 melanoma. Moreover, compared with the control hydrogel without D1MT, the immune‐stimulating hydrogel showed better efficiency in improving the immunosuppressive TME with increased number of activated T cells and reduced number of Tregs. Therefore, the immune‐stimulating hydrogel has great potential as a stimuli‐responsive platform for regulating suppressive TME and enhanced anti‐tumor chemo‐immunotherapy.
Distant metastasis is a primary cause of mortality and contributes to poor surgical outcomes in cancer patients. Before the development of organ-specific metastasis, the formation of a pre-metastatic niche is pivotal in promoting the spread of cancer cells. This review delves into the intricate landscape of the pre-metastatic niche, focusing on the roles of tumor-derived secreted factors, extracellular vesicles, and circulating tumor cells in shaping the metastatic niche. The discussion encompasses cellular elements such as macrophages, neutrophils, bone marrow-derived suppressive cells, and T/B cells, in addition to molecular factors like secreted substances from tumors and extracellular vesicles, within the framework of pre-metastatic niche formation. Insights into the temporal mechanisms of pre-metastatic niche formation such as epithelial-mesenchymal transition, immunosuppression, extracellular matrix remodeling, metabolic reprogramming, vascular permeability and angiogenesis are provided. Furthermore, the landscape of pre-metastatic niche in different metastatic organs like lymph nodes, lungs, liver, brain, and bones is elucidated. Therapeutic approaches targeting the cellular and molecular components of pre-metastatic niche, as well as interventions targeting signaling pathways such as the TGF-β, VEGF, and MET pathways, are highlighted. This review aims to enhance our understanding of pre-metastatic niche dynamics and provide insights for developing effective therapeutic strategies to combat tumor metastasis.
Adoptive immunotherapy, notably involving chimeric antigen receptor (CAR)-T cells, has obtained Food and Drug Administration (FDA) approval as a treatment for various hematological malignancies, demonstrating promising preclinical efficacy against cancers. However, the intricate and resource-intensive autologous cell processing, encompassing collection, expansion, engineering, isolation, and administration, hamper the efficacy of this therapeutic modality. Furthermore, conventional CAR T therapy is presently confined to addressing solid tumors due to impediments posed by physical barriers, the potential for cytokine release syndrome, and cellular exhaustion induced by the immunosuppressive and heterogeneous tumor microenvironment. Consequently, a strategic integration of adoptive immunotherapy with synergistic multimodal treatments, such as chemotherapy, radiotherapy, and vaccine therapy etc., emerges as a pivotal approach to surmount these inherent challenges. This collaborative strategy holds the key to addressing the limitations delineated above, thereby facilitating the realization of more precise personalized therapies characterized by heightened therapeutic efficacy. Such synergistic strategy not only serves to mitigate the constraints associated with adoptive immunotherapy but also fosters enhanced clinical applicability, thereby advancing the frontiers of therapeutic precision and effectiveness.
Adoptive cell therapy (ACT) is on the horizon as a thrilling therapeutic plan for cancer. However, widespread application of ACT is often restricted by several challenges, including complexity of priming tumor-specific T cells and poor trafficking in solid tumors. The convergence of nanotechnology and cancer immunotherapy is coming of age and could address the limitations of ACT. Recent studies have provided evidence on the application of magnetic nanoparticles (MNPs) to generate smart immune cells and to bypass problems associated with conventional ACT. Herein, we review current progress in the application of MNPs to improve preparing, guiding and tracking immune cells in cancer ACT. Besides, we comment on the challenges ahead and strategies to optimize MNPs for clinical settings.
CRISPR takes first steps in humans
CRISPR-Cas9 is a revolutionary gene-editing technology that offers the potential to treat diseases such as cancer, but the effects of CRISPR in patients are currently unknown. Stadtmauer et al. report a phase 1 clinical trial to assess the safety and feasibility of CRISPR-Cas9 gene editing in three patients with advanced cancer (see the Perspective by Hamilton and Doudna). They removed immune cells called T lymphocytes from patients and used CRISPR-Cas9 to disrupt three genes ( TRAC, TRBC , and PDCD1 ) with the goal of improving antitumor immunity. A cancer-targeting transgene, NY-ESO-1, was also introduced to recognize tumors. The engineered cells were administered to patients and were well tolerated, with durable engraftment observed for the study duration. These encouraging observations pave the way for future trials to study CRISPR-engineered cancer immunotherapies.
Science , this issue p. eaba7365 ; see also p. 976
T cell exhaustion limits immune responses against cancer and is a major cause of resistance to CAR- T cell therapeutics. Using a model wherein tonic CAR signaling induces hallmark features of exhaustion, we employed a drug-regulatable CAR to test the impact of transient cessation of receptor signaling (i.e. "rest") on the development and maintenance of exhaustion. Induction of rest in exhausting or already-exhausted CAR-T cells resulted in acquisition of a memory-like phenotype, improved anti-tumor functionality, and wholescale transcriptional and epigenetic reprogramming. Similar results were achieved with the Src kinase inhibitor dasatinib, which reversibly suppresses CAR signaling. The degree of functional reinvigoration was proportional to the duration of rest and was associated with expression of transcription factors TCF1 and LEF1. This work demonstrates that transient cessation of CAR-T cell signaling can enhance anti-tumor potency by preventing or reversing exhaustion and challenges the paradigm that exhaustion is an epigenetically fixed state.
Chimeric antigen receptor (CAR) T cells mediate anti-tumour effects in a small subset of patients with cancer1,2,3, but dysfunction due to T cell exhaustion is an important barrier to progress4,5,6. To investigate the biology of exhaustion in human T cells expressing CAR receptors, we used a model system with a tonically signaling CAR, which induces hallmark features of exhaustion⁶. Exhaustion was associated with a profound defect in the production of IL-2, along with increased chromatin accessibility of AP-1 transcription factor motifs and overexpression of the bZIP and IRF transcription factors that have been implicated in mediating dysfunction in exhausted T cells7,8,9,10. Here we show that CAR T cells engineered to overexpress the canonical AP-1 factor c-Jun have enhanced expansion potential, increased functional capacity, diminished terminal differentiation and improved anti-tumour potency in five different mouse tumour models in vivo. We conclude that a functional deficiency in c-Jun mediates dysfunction in exhausted human T cells, and that engineering CAR T cells to overexpress c-Jun renders them resistant to exhaustion, thereby addressing a major barrier to progress for this emerging class of therapeutic agents.
TCR-gene-transfer is an efficient strategy to produce therapeutic T cells of defined antigen specificity. However, there are substantial variations in the cell surface expression levels of human TCRs, which can impair the function of engineered T cells. Here we demonstrate that substitutions of 3 amino acid residues in the framework of the TCR variable domains consistently increase the expression of human TCRs on the surface of engineered T cells.The modified TCRs mediate enhanced T cell proliferation, cytokine production and cytotoxicity, while reducing the peptide concentration required for triggering effector function up to 3000-fold. Adoptive transfer experiments in mice show that modified TCRs control tumor growth more efficiently than wild-type TCRs. Our data indicate that simple variable domain modifications at a distance from the antigen-binding loops lead to increased TCR expression and improved effector function. This finding provides a generic platform to optimize the efficacy of TCR gene therapy in humans.
Fibrosis is observed in nearly every form of myocardial disease¹. Upon injury, cardiac fibroblasts in the heart begin to remodel the myocardium by depositing excess extracellular matrix, resulting in increased stiffness and reduced compliance of the tissue. Excessive cardiac fibrosis is an important factor in the progression of various forms of cardiac disease and heart failure². However, clinical interventions and therapies that target fibrosis remain limited³. Here we demonstrate the efficacy of redirected T cell immunotherapy to specifically target pathological cardiac fibrosis in mice. We find that cardiac fibroblasts that express a xenogeneic antigen can be effectively targeted and ablated by adoptive transfer of antigen-specific CD8⁺ T cells. Through expression analysis of the gene signatures of cardiac fibroblasts obtained from healthy and diseased human hearts, we identify an endogenous target of cardiac fibroblasts—fibroblast activation protein. Adoptive transfer of T cells that express a chimeric antigen receptor against fibroblast activation protein results in a significant reduction in cardiac fibrosis and restoration of function after injury in mice. These results provide proof-of-principle for the development of immunotherapeutic drugs for the treatment of cardiac disease.
Chimeric antigen receptor (CAR)-T-cell therapy for solid tumors is limited due to heterogeneous target antigen expression and outgrowth of tumors lacking the antigen targeted by CAR-T cells directed against single antigens. Here, we developed a bicistronic construct to drive expression of a CAR specific for EGFRvIII, a glioblastoma-specific tumor antigen, and a bispecific T-cell engager (BiTE) against EGFR, an antigen frequently overexpressed in glioblastoma but also expressed in normal tissues. CART.BiTE cells secreted EGFR-specific BiTEs that redirect CAR-T cells and recruit untransduced bystander T cells against wild-type EGFR. EGFRvIII-specific CAR-T cells were unable to completely treat tumors with heterogenous EGFRvIII expression, leading to outgrowth of EGFRvIII-negative, EGFR-positive glioblastoma. However, CART.BiTE cells eliminated heterogenous tumors in mouse models of glioblastoma. BiTE-EGFR was locally effective but was not detected systemically after intracranial delivery of CART.BiTE cells. Unlike EGFR-specific CAR-T cells, CART.BiTE cells did not result in toxicity against human skin grafts in vivo. BiTE-secreting CAR-T cells overcome antigen escape from EGFRvIII-targeted therapy for glioblastoma.
Cancer arises from the accumulation of genetic alterations, which can lead to the production of mutant proteins not expressed by normal cells. These mutant proteins can be processed and presented on the cell surface by major histocompatibility complex molecules as neoepitopes, allowing CD8 + T cells to mount responses against them. For solid tumors, only an average 2% of neoepitopes predicted by algorithms have detectable endogenous antitumor T cell responses. This suggests that low mutation burden tumors, which include many pediatric tumors, are poorly immunogenic. Here, we report that pediatric patients with acute lymphoblastic leukemia (ALL) have tumor-associated neoepitope-specific CD8 + T cells, responding to 86% of tested neoantigens and recognizing 68% of the tested neoepitopes. These responses include a public neoantigen from the ETV6-RUNX1 fusion that is targeted in seven of nine tested patients. We characterized phenotypic and transcriptional profiles of CD8 + tumor-infiltrating lympho-cytes (TILs) at the single-cell level and found a heterogeneous population that included highly functional effectors. Moreover, we observed immunodominance hierarchies among the CD8 + TILs restricted to one or two putative neoepitopes. Our results indicate that robust antitumor immune responses are induced in pediatric ALL despite their low mutation burdens and emphasize the importance of immunodominance in shaping cellular immune responses. Furthermore, these data suggest that pediatric cancers may be amenable to immunotherapies aimed at enhancing immune recognition of tumor-specific neoantigens.
Relapse after allogeneic hematopoietic cell transplantation (HCT) is the leading cause of death in patients with acute myeloid leukemia (AML) entering HCT with poor-risk features1–3. When HCT does produce prolonged relapse-free survival, it commonly reflects graft-versus-leukemia effects mediated by donor T cells reactive with antigens on leukemic cells⁴. As graft T cells have not been selected for leukemia specificity and frequently recognize proteins expressed by many normal host tissues, graft-versus-leukemia effects are often accompanied by morbidity and mortality from graft-versus-host disease⁵. Thus, AML relapse risk might be more effectively reduced with T cells expressing receptors (TCRs) that target selected AML antigens⁶. We therefore isolated a high-affinity Wilms’ Tumor Antigen 1-specific TCR (TCRC4) from HLA-A2⁺ normal donor repertoires, inserted TCRC4 into Epstein–Bar virus-specific donor CD8⁺ T cells (TTCR-C4) to minimize graft-versus-host disease risk and enhance transferred T cell survival7,8, and infused these cells prophylactically post-HCT into 12 patients (NCT01640301). Relapse-free survival was 100% at a median of 44 months following infusion, while a concurrent comparative group of 88 patients with similar risk AML had 54% relapse-free survival (P = 0.002). TTCR-C4 maintained TCRC4 expression, persisted long-term and were polyfunctional. This strategy appears promising for preventing AML recurrence in individuals at increased risk of post-HCT relapse.
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Background: Anti-CD19 CAR-T cell therapy is a breakthrough treatment (tx) for patients (pts) with relapsed/refractory (R/R) B-cell non-Hodgkin lymphoma (NHL). Despite impressive outcomes, non-response and relapse with CD19 negative disease remain challenges. Through dual B-cell antigen targeting of CD20 and CD19, with a first-in-human bispecific lentiviral CAR-T cell (LV20.19CAR), we aim to improve response rates while limiting CD19 negative relapse. Methods: Pts were treated on a Phase 1 dose escalation + expansion trial (NCT03019055) to demonstrate safety of a 41BB/CD3z LV20.19CAR T cell for adults with R/R B-cell NHL. Safety was assessed by incidence of dose limiting toxicities (DLTs) within 28 days post-infusion. Starting dose was 2.5 x 10^5 cells/kg with a target dose of 2.5 x 10^6 cells/kg. All pts received fludarabine+cyclophosphamide for lymphodepletion. Results: 11 pts have completed tx to date. 9 pts in dose escalation and 2 pts in expansion phase. Median age was 54 years (46-67) and histology included DLBCL = 5 pts, MCL = 4 pts, and CLL = 2 pts. In dose escalation, 3 pts were treated at 2.5 x 10^5 cells/kg, 3 pts at 7.5 x 10^5 cells/kg, and 3 pts at 2.5 x 10^6 cells/kg with no DLTs. As a result, 2.5 x 10^6 cells/kg was selected for expansion. In terms of safety, 6 pts developed Grade 1-2 cytokine release syndrome (CRS) and 3 pts had Grade 1-2 neurotoxicity (NTX). No patient had grade 3-4 CRS or NTX and none required ICU level care. 4 pts required 1-2 doses of tocilizumab for CRS. The day 28 overall response rate (ORR) for all pts was 82% (6/11 = complete response (CR) and 3/11 = partial response). All CR pts remain in remission, the longest > 1 year. All progressing pts underwent repeat biopsy, and all retained either CD19 or CD20 positivity. Additional pts are being enrolled in the expansion phase and updated data will be presented. Conclusions: Phase 1 results from the LV20.19 CAR T clinical trial demonstrate that infusion of 2.5 x 10^6 cells/kg is safe for further investigation with no DLTs among treated pts. Down-regulation of target antigens was not identified as a mechanism of resistance in progressing pts. With limited toxicity and encouraging ORR, dual targeted LV20.19CAR T cells merits further investigation. Clinical trial information: NCT03019055.
Purpose:
Chimeric antigen receptor T-cell (CART) therapy targeting CD22 induces remission in 70% of patients with relapsed/refractory acute lymphoblastic leukemia (ALL). However, the majority of post-CD22 CART remissions are short and associated with reduction in CD22 expression. We evaluate the implications of low antigen density on the activity of CD22 CART and propose mechanisms to overcome antigen escape.
Experimental design:
Using ALL cell lines with variable CD22 expression, we evaluate the cytokine profile, cytotoxicity, and in vivo CART functionality in the setting of low CD22 expression. We develop a high-affinity CD22 chimeric antigen receptor (CAR) as an approach to improve CAR sensitivity. We also assess Bryostatin1, a therapeutically relevant agent, to upregulate CD22 and improve CAR functionality.
Results:
We demonstrate that low CD22 expression negatively impacts in vitro and in vivo CD22 CART functionality and impairs in vivo CART persistence. Moreover, low antigen expression on leukemic cells increases naïve phenotype of persisting CART. Increasing CAR affinity does not improve response to low-antigen leukemia. Bryostatin1 upregulates CD22 on leukemia and lymphoma cell lines for 1 week following single-dose exposure, and improves CART functionality and in vivo persistence. While Bryostatin1 attenuates IFNγ production by CART, overall in vitro and in vivo CART cytotoxicity is not adversely affected. Finally, administration of Bryostain1 with CD22 CAR results in longer duration of in vivo response.
Conclusions:
We demonstrate that target antigen modulation is a promising strategy to improve CD22 CAR efficacy and remission durability in patients with leukemia and lymphoma.See related commentary by Guedan and Delgado, p. 5188.
Background:
Chimeric antigen receptor (CAR) T cells can induce remission in highly refractory leukemia and lymphoma subjects, yet the parameters for achieving sustained relapse-free survival are not fully delineated.
Methods:
We analyzed 43 pediatric and young adult subjects participating in a Phase I trial of defined composition CD19CAR T cells (NCT02028455). CAR T cell phenotype, function and expansion, as well as starting material T cell repertoire, were analyzed in relation to therapeutic outcome (defined as achieving complete remission within 63 days) and duration of leukemia free survival and B cell aplasia.
Results:
These analyses reveal that initial therapeutic failures (n = 5) were associated with attenuated CAR T cell expansion and/or rapid attrition of functional CAR effector cells following adoptive transfer. The CAR T products were similar in phenotype and function when compared to products resulting in sustained remissions. However, the initial apheresed peripheral blood T cells could be distinguished by an increased frequency of LAG-3+/TNF-αlow CD8 T cells and, following adoptive transfer, the rapid expression of exhaustion markers. For the 38 subjects who achieved an initial sustained MRD-neg remission, remission durability correlated with therapeutic products having increased frequencies of TNF-α-secreting CAR CD8+ T cells, and was dependent on a sufficiently high CD19+ antigen load at time of infusion to trigger CAR T cell proliferation.
Conclusion:
These parameters have the potential to prospectively identify patients at risk for therapeutic failure and support the development of approaches to boost CAR T cell activation and proliferation in patients with low levels of CD19 antigen.
Trial registration:
ClinicalTrials.gov NCT02028455.
Funding:
Partial funding for this study was provided by Stand Up to Cancer & St. Baldrick's Pediatric Dream Team Translational Research Grant (SU2C-AACR-DT1113), RO1 CA136551-05, Alex Lemonade Stand Phase I/II Infrastructure Grant, Conquer Cancer Foundation Career Development Award, Washington State Life Sciences Discovery Fund, Ben Towne Foundation, William Lawrence & Blanche Hughes Foundation, and Juno Therapeutics, Inc., a Celgene Company.
T cells expressing chimeric antigen receptors (CAR T cells) targeting human CD19 (hCD19) have shown clinical efficacy against B cell malignancies1,2. CAR T cells have been less effective against solid tumours3–5, in part because they enter a hyporesponsive (‘exhausted’ or ‘dysfunctional’) state6–9 triggered by chronic antigen stimulation and characterized by upregulation of inhibitory receptors and loss of effector function. To investigate the function of CAR T cells in solid tumours, we transferred hCD19-reactive CAR T cells into hCD19⁺ tumour-bearing mice. CD8⁺CAR⁺ tumour-infiltrating lymphocytes and CD8⁺ endogenous tumour-infiltrating lymphocytes expressing the inhibitory receptors PD-1 and TIM3 exhibited similar profiles of gene expression and chromatin accessibility, associated with secondary activation of nuclear receptor transcription factors NR4A1 (also known as NUR77), NR4A2 (NURR1) and NR4A3 (NOR1) by the initiating transcription factor NFAT (nuclear factor of activated T cells)10–12. CD8⁺ T cells from humans with cancer or chronic viral infections13–15 expressed high levels of NR4A transcription factors and displayed enrichment of NR4A-binding motifs in accessible chromatin regions. CAR T cells lacking all three NR4A transcription factors (Nr4a triple knockout) promoted tumour regression and prolonged the survival of tumour-bearing mice. Nr4a triple knockout CAR tumour-infiltrating lymphocytes displayed phenotypes and gene expression profiles characteristic of CD8⁺ effector T cells, and chromatin regions uniquely accessible in Nr4a triple knockout CAR tumour-infiltrating lymphocytes compared to wild type were enriched for binding motifs for NF-κB and AP-1, transcription factors involved in activation of T cells. We identify NR4A transcription factors as having an important role in the cell-intrinsic program of T cell hyporesponsiveness and point to NR4A inhibition as a promising strategy for cancer immunotherapy.
Autologous induced pluripotent stem cells (iPSCs) constitute an unlimited cell source for patient-specific cell-based organ repair strategies. However, their generation and subsequent differentiation into specific cells or tissues entail cell line-specific manufacturing challenges and form a lengthy process that precludes acute treatment modalities. These shortcomings could be overcome by using prefabricated allogeneic cell or tissue products, but the vigorous immune response against histo-incompatible cells has prevented the successful implementation of this approach. Here we show that both mouse and human iPSCs lose their immunogenicity when major histocompatibility complex (MHC) class I and II genes are inactivated and CD47 is over-expressed. These hypoimmunogenic iPSCs retain their pluripotent stem cell potential and differentiation capacity. Endothelial cells, smooth muscle cells, and cardiomyocytes derived from hypoimmunogenic mouse or human iPSCs reliably evade immune rejection in fully MHC-mismatched allogeneic recipients and survive long-term without the use of immunosuppression. These findings suggest that hypoimmunogenic cell grafts can be engineered for universal transplantation. © 2019, The Author(s), under exclusive licence to Springer Nature America, Inc.
Across clinical trials, T cell expansion and persistence following adoptive cell transfer (ACT) have correlated with superior patient outcomes. Herein, we undertook a pan-cancer analysis to identify actionable ligand-receptor pairs capable of compromising T cell durability following ACT. We discovered that FASLG, the gene encoding the apoptosis-inducing ligand FasL, is overexpressed within the majority of human tumor microenvironments (TMEs). Further, we uncovered that Fas, the receptor for FasL, is highly expressed on patient-derived T cells used for clinical ACT. We hypothesized that a cognate Fas-FasL interaction within the TME might limit both T cell persistence and antitumor efficacy. We discovered that genetic engineering of Fas variants impaired in the ability to bind FADD functioned as dominant negative receptors (DNRs), preventing FasL-induced apoptosis in Fas-competent T cells. T cells coengineered with a Fas DNR and either a T cell receptor or chimeric antigen receptor exhibited enhanced persistence following ACT, resulting in superior antitumor efficacy against established solid and hematologic cancers. Despite increased longevity, Fas DNR-engineered T cells did not undergo aberrant expansion or mediate autoimmunity. Thus, T cell-intrinsic disruption of Fas signaling through genetic engineering represents a potentially universal strategy to enhance ACT efficacy across a broad range of human malignancies.
Purpose:
Patients with relapsed pediatric solid tumors and CNS malignancies have few therapeutic options and frequently die of their disease. Chimeric antigen receptor (CAR) T cells have shown tremendous success in treating relapsed pediatric acute lymphoblastic leukemia, but this has not yet translated to treating solid tumors. This is partially due to a paucity of differentially expressed cell surface molecules on solid tumors that can be safely targeted. Here, we present B7-H3 (CD276) as a putative target for CAR T-cell therapy of pediatric solid tumors, including those arising in the central nervous system.
Experimental design:
We developed a novel B7-H3 CAR whose binder is derived from a mAb that has been shown to preferentially bind tumor tissues and has been safely used in humans in early-phase clinical trials. We tested B7-H3 CAR T cells in a variety of pediatric cancer models.
Results:
B7-H3 CAR T cells mediate significant antitumor activity in vivo, causing regression of established solid tumors in xenograft models including osteosarcoma, medulloblastoma, and Ewing sarcoma. We demonstrate that B7-H3 CAR T-cell efficacy is largely dependent upon high surface target antigen density on tumor tissues and that activity is greatly diminished against target cells that express low levels of antigen, thus providing a possible therapeutic window despite low-level normal tissue expression of B7-H3.
Conclusions:
B7-H3 CAR T cells could represent an exciting therapeutic option for patients with certain lethal relapsed or refractory pediatric malignancies, and should be tested in carefully designed clinical trials.
Background:
Axicabtagene ciloleucel is an autologous anti-CD19 chimeric antigen receptor (CAR) T-cell therapy. In the previous analysis of the ZUMA-1 registrational study, with a median follow-up of 15·4 months (IQR 13·7-17·3), 89 (82%) of 108 assessable patients with refractory large B-cell lymphoma treated with axicabtagene ciloleucel achieved an objective response, and complete responses were noted in 63 (58%) patients. Here we report long-term activity and safety outcomes of the ZUMA-1 study.
Methods:
ZUMA-1 is a single-arm, multicentre, registrational trial at 22 sites in the USA and Israel. Eligible patients were aged 18 years or older, and had histologically confirmed large B-cell lymphoma-including diffuse large B-cell lymphoma, primary mediastinal B-cell lymphoma, and transformed follicular lymphoma-according to the 2008 WHO Classification of Tumors of Hematopoietic and Lymphoid Tissue; refractory disease or relapsed after autologous stem-cell transplantation; an Eastern Cooperative Oncology Group performance status of 0 or 1; and had previously received an anti-CD20 monoclonal antibody containing-regimen and an anthracycline-containing chemotherapy. Participants received one dose of axicabtagene ciloleucel on day 0 at a target dose of 2 × 106 CAR T cells per kg of bodyweight after conditioning chemotherapy with intravenous fludarabine (30 mg/m2 body-surface area) and cyclophosphamide (500 mg/m2 body-surface area) on days -5, -4, and -3. The primary endpoints were safety for phase 1 and the proportion of patients achieving an objective response for phase 2, and key secondary endpoints were overall survival, progression-free survival, and duration of response. Pre-planned activity and safety analyses were done per protocol. ZUMA-1 is registered with ClinicalTrials.gov, number NCT02348216. Although the registrational cohorts are closed, the trial remains open, and recruitment to extension cohorts with alternative endpoints is underway.
Findings:
Between May 19, 2015, and Sept 15, 2016, 119 patients were enrolled and 108 received axicabtagene ciloleucel across phases 1 and 2. As of the cutoff date of Aug 11, 2018, 101 patients assessable for activity in phase 2 were followed up for a median of 27·1 months (IQR 25·7-28·8), 84 (83%) had an objective response, and 59 (58%) had a complete response. The median duration of response was 11·1 months (4·2-not estimable). The median overall survival was not reached (12·8-not estimable), and the median progression-free survival was 5·9 months (95% CI 3·3-15·0). 52 (48%) of 108 patients assessable for safety in phases 1 and 2 had grade 3 or worse serious adverse events. Grade 3 or worse cytokine release syndrome occurred in 12 (11%) patients, and grade 3 or worse neurological events in 35 (32%). Since the previous analysis at 1 year, additional serious adverse events were reported in four patients (grade 3 mental status changes, grade 4 myelodysplastic syndrome, grade 3 lung infection, and two episodes of grade 3 bacteraemia), none of which were judged to be treatment related. Two treatment-related deaths (due to haemophagocytic lymphohistiocytosis and cardiac arrest) were previously reported, but no new treatment-related deaths occurred during the additional follow-up.
Interpretation:
These 2-year follow-up data from ZUMA-1 suggest that axicabtagene ciloleucel can induce durable responses and a median overall survival of greater than 2 years, and has a manageable long-term safety profile in patients with relapsed or refractory large B-cell lymphoma.
Funding:
Kite and the Leukemia & Lymphoma Society Therapy Acceleration Program.
Despite high remission rates following CAR-T cell therapy in B-ALL, relapse due to loss of the targeted antigen is increasingly recognized as a mechanism of immune escape. We hypothesized that simultaneous targeting of CD19 and CD22 may reduce the likelihood of antigen loss, thus improving sustained remission rates. A systematic approach to the generation of CAR constructs incorporating two target-binding domains led to several novel CD19/CD22 bivalent CAR constructs. Importantly, we demonstrate the challenges associated with the construction of a bivalent CAR format that preserves bifunctionality against both CD19 and CD22. Using the most active bivalent CAR constructs, we found similar transduction efficiency compared to that of either CD19 or CD22 single CARs alone. When expressed on human T cells, the optimized CD19/CD22 CAR construct induced comparable interferon γ and interleukin-2 in vitro compared to single CARs against dual-antigen-expressing as well as single-antigen-expressing cell lines. Finally, the T cells expressing CD19/CD22 CAR eradicated ALL cell line xenografts and patient-derived xenografts (PDX), including a PDX generated from a patient with CD19⁻ relapse following CD19-directed CAR therapy. The CD19/CD22 bivalent CAR provides an opportunity to test whether simultaneous targeting may reduce risk of antigen loss.
Adoptive cell transfer (ACT) of tumor-infiltrating lymphocytes (TILs) targeting neoantigens can mediate tumor regression in selected patients with metastatic epithelial cancer. However, effectively identifying and harnessing neoantigen-reactive T cells for patient treatment remains a challenge and it is unknown whether current methods to detect neoantigen-reactive T cells are missing potentially clinically relevant neoantigen reactivities. We thus investigated whether the detection of neoantigen-reactive TILs could be enhanced by enriching T cells that express PD-1 and/or T cell activation markers followed by microwell culturing to avoid overgrowth of nonreactive T cells. In 6 patients with metastatic epithelial cancer, this method led to the detection of CD4+ and CD8+ T cells targeting 18 and 1 neoantigens, respectively, compared with 6 and 2 neoantigens recognized by CD4+ and CD8+ T cells, respectively, when using our standard TIL fragment screening approach. In 2 patients, no recognition of mutated peptides was observed using our conventional screen, while our high-throughput approach led to the identification of 5 neoantigen-reactive T cell receptors (TCRs) against 5 different mutations from one patient and a highly potent MHC class II-restricted KRASG12V-reactive TCR from a second patient. In addition, in a metastatic tumor sample from a patient with serous ovarian cancer, we isolated 3 MHC class II-restricted TCRs targeting the TP53G245S hot-spot mutation. In conclusion, this approach provides a highly sensitive platform to isolate clinically relevant neoantigen-reactive T cells or their TCRs for cancer treatment.
The efficacy of chimeric antigen receptor (CAR) T cell therapy against poorly responding tumors can be enhanced by administering the cells in combination with immune checkpoint blockade inhibitors. Alternatively, the CAR construct has been engineered to coexpress factors that boost CAR-T cell function in the tumor microenvironment. We modified CAR-T cells to secrete PD-1-blocking single-chain variable fragments (scFv). These scFv-secreting CAR-T cells acted in both a paracrine and autocrine manner to improve the anti-tumor activity of CAR-T cells and bystander tumor-specific T cells in clinically relevant syngeneic and xenogeneic mouse models of PD-L1+ hematologic and solid tumors. The efficacy was similar to or better than that achieved by combination therapy with CAR-T cells and a checkpoint inhibitor. This approach may improve safety, as the secreted scFvs remained localized to the tumor, protecting CAR-T cells from PD-1 inhibition, which could potentially avoid toxicities associated with systemic checkpoint inhibition.
Chimeric antigen receptor (CAR) T cells have emerged as a novel form of treatment of patients with B-cell malignancies. In particular, anti-CD19 CAR T-cell therapy has effected impressive clinical responses in B-cell acute lymphoblastic leukemia and diffuse large B-cell lymphoma. However, not all patients respond, and relapse with antigen loss has been observed in all patient subsets. Here, we report on the design and optimization of a novel CAR directed to the surface antigen CD37, which is expressed in B-cell non-Hodgkin lymphomas, in chronic lymphocytic leukemia, and in some cases of cutaneous and peripheral T-cell lymphomas. We found that CAR-37 T cells demonstrated antigen-specific activation, cytokine production, and cytotoxic activity in models of B- and T-cell lymphomas in vitro and in vivo, including patient-derived xenografts. Taken together, these results are the first showing that T cells expressing anti-CD37 CAR have substantial activity against 2 different lymphoid lineages, without evidence of significant T-cell fratricide. Furthermore, anti-CD37 CARs were readily combined with anti-CD19 CARs to generate dual-specific CAR T cells capable of recognizing CD19 and CD37 alone or in combination. Our findings indicate that CD37-CAR T cells represent a novel therapeutic agent for the treatment of patients with CD37-expressing lymphoid malignancies.
Insufficient reactivity against cells with low antigen density has emerged as an important cause of chimeric antigen receptor (CAR) T-cell resistance. Little is known about factors that modulate the threshold for antigen recognition. We demonstrate that CD19 CAR activity is dependent upon antigen density and that the CAR construct in axicabtagene ciloleucel (CD19-CD28ζ) outperforms that in tisagenlecleucel (CD19-4-1BBζ) against antigen-low tumors. Enhancing signal strength by including additional immunoreceptor tyrosine-based activation motifs (ITAM) in the CAR enables recognition of low-antigen-density cells, whereas ITAM deletions blunt signal and increase the antigen density threshold. Furthermore, replacement of the CD8 hinge-transmembrane (H/T) region of a 4-1BBζ CAR with a CD28-H/T lowers the threshold for CAR reactivity despite identical signaling molecules. CARs incorporating a CD28-H/T demonstrate a more stable and efficient immunologic synapse. Precise design of CARs can tune the threshold for antigen recognition and endow 4-1BBζ-CARs with enhanced capacity to recognize antigen-low targets while retaining a superior capacity for persistence.
Significance
Optimal CAR T-cell activity is dependent on antigen density, which is variable in many cancers, including lymphoma and solid tumors. CD28ζ-CARs outperform 4-1BBζ-CARs when antigen density is low. However, 4-1BBζ-CARs can be reengineered to enhance activity against low-antigen-density tumors while maintaining their unique capacity for persistence.
This article is highlighted in the In This Issue feature, p. 627
Background:
Anti-CD19 chimeric antigen receptor (CAR) T-cell therapy has shown remarkable clinical efficacy in B-cell cancers. However, CAR T cells can induce substantial toxic effects, and the manufacture of the cells is complex. Natural killer (NK) cells that have been modified to express an anti-CD19 CAR have the potential to overcome these limitations.
Methods:
In this phase 1 and 2 trial, we administered HLA-mismatched anti-CD19 CAR-NK cells derived from cord blood to 11 patients with relapsed or refractory CD19-positive cancers (non-Hodgkin's lymphoma or chronic lymphocytic leukemia [CLL]). NK cells were transduced with a retroviral vector expressing genes that encode anti-CD19 CAR, interleukin-15, and inducible caspase 9 as a safety switch. The cells were expanded ex vivo and administered in a single infusion at one of three doses (1×105, 1×106, or 1×107 CAR-NK cells per kilogram of body weight) after lymphodepleting chemotherapy.
Results:
The administration of CAR-NK cells was not associated with the development of cytokine release syndrome, neurotoxicity, or graft-versus-host disease, and there was no increase in the levels of inflammatory cytokines, including interleukin-6, over baseline. The maximum tolerated dose was not reached. Of the 11 patients who were treated, 8 (73%) had a response; of these patients, 7 (4 with lymphoma and 3 with CLL) had a complete remission, and 1 had remission of the Richter's transformation component but had persistent CLL. Responses were rapid and seen within 30 days after infusion at all dose levels. The infused CAR-NK cells expanded and persisted at low levels for at least 12 months.
Conclusions:
Among 11 patients with relapsed or refractory CD19-positive cancers, a majority had a response to treatment with CAR-NK cells without the development of major toxic effects. (Funded by the M.D. Anderson Cancer Center CLL and Lymphoma Moonshot and the National Institutes of Health; ClinicalTrials.gov number, NCT03056339.).
Many potential targets for CAR-T cells in solid tumors are expressed in some normal tissues, raising concern for off-tumor toxicity. Following lymphodepletion, CAR-T cells targeting the tumor-associated antigen ROR1 lysed tumors in mice but induced lethal bone marrow failure due to recognition of ROR1+ stromal cells. To improve selectivity, we engineered T cells with synthetic Notch (synNotch) receptors specific for EpCAM or B7-H3, which are expressed on ROR1+ tumor cells but not ROR1+ stromal cells. SynNotch receptors induced ROR1 CAR expression selectively within the tumor, resulting in tumor regression without toxicity when tumor cells were segregated from, but not when co-localized with, normal ROR1+ cells. This strategy, thus, permits safe targeting of tumors that are sufficiently separated from normal cells.
Key Points
A trimeric extracellular moiety of APRIL has enhanced binding to BCMA and TACI compared with monomeric APRIL when incorporated into a CAR. T cells transduced with a trimeric APRIL-based CAR are a promising approach for the treatment of MM.
Chimeric Antigen Receptor (CAR) therapy targeting CD19 achieves complete remission (CR) rates of 70%-90% in relapsed/refractory B-ALL. Relapse due to loss of the CD19 targeted epitope presents a therapeutic challenge as evidenced by the largest global pediatric CD19-CAR experience which showed 15 of 16 relapses to be explained by CD19 downregulation (Maude et al, NEJM 2018). Alternatively targeting CD22 using CD22-CAR therapy has demonstrated a CR rate of approximately 70% in both CD19+ and CD19- B-ALL, however relapse due to CD22 downregulation limits the curative potential of singularly-targeting CD22 (Fry et al, Nat Med. 2018). We hypothesized that simultaneous targeting of CD19 and CD22 via a bispecific CAR-T cell would be a safe and tolerable treatment strategy in relapsed/refractory B-cell ALL and address immune evasion. Here, we report the first clinical experience in pediatric patients using bispecific CD19-CD22 CAR T cells.
We describe a single institution phase I dose escalation study in pediatric patients with relapsed or refractory B cell ALL. We utilized lentiviral transduction of a bivalent CAR construct incorporating the fmc63 CD19 and m971 CD22 single chain variable fragments (scFvs) used in clinically tested CAR constructs and a 41BB costimulatory endodomain (Fry et al, Nat Med. 2018). Our primary objectives are feasibility of production of this bivalent CAR and safety at 3 dose escalation levels (1x106, 3x106 and 1x107 CAR T cells/kg). Clinical response assessment is evaluated as a secondary aim. All patients described received lymphodepletion with fludarabine (25mg/m2 x 3 days) and cyclophosphamide (900mg/m2 x 1) followed by fresh or cryopreserved CAR T cell infusion after a 7-9 day production time. Patients were prospectively monitored at predefined intervals for disease response and correlative assessments.
Four pediatric patients with precursor-B ALL, age 2-17, have been enrolled and treated with CD19/CD22 bispecific CAR T cells at dose level 1 (1x106) [Table 1]. Three patients entered CAR therapy with low disease burden detected by minimal residual disease (MRD) alone and 1 patient initiated therapy with 12% bone marrow blasts. All patients were CNS1 at time of treatment. The toxicity profile mirrored that of the singular CD19 and CD22 CAR experience with 3 patients experiencing reversible CRS (2 Grade I, 1 Grade II), onset day 3-8, and 2 patients experiencing grade I neurotoxicity, onset day 3-9. In our cohort, we experienced lower grade toxicities than previously reported, likely due to a mean lower disease burden. Only 1 patient with CRS met criteria for tocilizumab and this patient was the singular study patient treated with higher burden disease. Neurotoxicity was managed with supportive care and fully reversible. Peripheral blood flow cytometry analysis detects circulating CAR by day 6 in all patients and demonstrates peak CAR expansion between day 6-10. Peak CAR T expansion reached levels of 10-25% of total T cells with inter-patient variability in CD4 and CD8 predominance, favoring CD8 expansion in 3 of 4 patients. Clinical symptoms and inflammatory markers expectedly correlate with peak CAR expansion. Four of 4 patients achieved complete remission (CR) at day 28 post-CD19/CD22 bispecific CAR therapy. Three of 4 patients demonstrated MRD- remissions by flow cytometry and of these, next generation sequencing (NGS) was negative where available (N=2). Multi-parametric CyTOF studies permitting CAR T cell phenotyping in conjunction with single cell TCR tracking, proteomics, epigenomics and cytokine profiling are ongoing and will be used to further characterize persisting CAR T cells and define inter-product and inter-patient variability.
In this phase I study, we demonstrate safety and tolerability of this bispecific CD19/CD22 CAR at a dose of 1x106 CAR T cells/kg in pediatric patients with relapsed/refractory B cell ALL. The CD19/22-bispecific CAR mediated antileukemic activity in 100% of patients studied thus far. Long-term follow up and further accrual will be required to inform the effect of bispecific CAR targeting on surface antigen remodeling.
Disclosures
Muffly: Adaptive Biotechnologies: Research Funding; Shire Pharmaceuticals: Research Funding. Miklos:Genentech: Research Funding; Kite - Gilead: Consultancy, Research Funding; Janssen: Consultancy, Research Funding; Pharmacyclics - Abbot: Consultancy, Research Funding; Adaptive Biotechnologies: Consultancy, Research Funding; Novartis: Consultancy, Research Funding.
Three patients with hematologic relapse after bone marrow transplantation for chronic myelogenous leukemia were treated with interferon alpha and transfusion of viable donor buffy coat. All had complete hematologic and cytogenetic remission, which persisted 32 to 91 weeks after treatment. In two patients graft-versus-host disease developed and was treated by immunosuppression. These results are an example of adoptive immunotherapy without cytoreductive chemotherapy or radiotherapy in human chimeras.
Chimeric Antigen Receptors (CARs) are engineered transmembrane proteins consisting of an antibody (ab)-derived antigen recognition domain linked to intracellular T cell signaling domains. Cytotoxic T cells endowed with tumor-reactive CARs are highly promising tools for immunotherapy of cancer. There are however only a few truly tumor specific molecules that can be targeted by CARs, a drawback for the broad application of CAR T cell therapy. Indeed, when we recently aimed at targeting CD38high multiple myeloma (MM) with T cells transduced with high affinity CD38CARs, we observed that they not only lysed the CD38high MM cells but also CD38+ normal hematopoietic cells, pointing towards potential safety issues of such tumor-associated, but not entirely tumor-specific CARs. Therefore, using CD38 as a model for antigen we now tested whether it would be possible to reduce the on target, off-tumor effects of such CARs by optimizing their target cell affinity. To this end, we generated a new panel of CD38 abs through the "light chain exchange" method, in which heavy chains of two high affinity CD38 abs were combined with 176 different germ line light chains. This approach revealed around 100 new abs, which displayed 10- >1000 fold lower affinity to CD38 as compared to the parental abs. After categorizing them in three classes based on CD38 binding affinity, we used 8 abs from each class to generate 24 different CD38-CAR constructs. Testing the cytotoxic activity of T cells transduced with these CD38-CARs against CD38++ MM cell lines, primary MM cells and CD38+ normal hematopoietic cells in vitro and in vivo demonstrated that CD38-CAR T cells with ca. 1000 fold lower affinity to CD38 could still effectively lyse CD38++ MM cells while there was little or no cytotoxicity against CD38+ healthy hematopoietic cells. The results of this study reveal that it is possible to reduce the on-target off-tumor effects of CARs by optimizing their affinity. Thus, tailored affinity of the ab binding domain may open up new roads for CAR therapy.
Disclosures
van de Donk: Janssen: Research Funding; Celgene: Research Funding; BMS: Research Funding; Amgen: Research Funding. Lokhorst:Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Genmab: Research Funding. Mutis:Celgene: Research Funding; Genmab: Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding.
‘T cell exhaustion’ is a broad term that has been used to describe the response of T cells to chronic antigen stimulation, first in the setting of chronic viral infection but more recently in response to tumours. Understanding the features of and pathways to exhaustion has crucial implications for the success of checkpoint blockade and adoptive T cell transfer therapies. In this Viewpoint article, 18 experts in the field tell us what exhaustion means to them, ranging from complete lack of effector function to altered functionality to prevent immunopathology, with potential differences between cancer and chronic infection. Their responses highlight the dichotomy between terminally differentiated exhausted T cells that are TCF1– and the self-renewing TCF1⁺ population from which they derive. These TCF1⁺ cells are considered by some to have stem cell-like properties akin to memory T cell populations, but the developmental relationships are unclear at present. Recent studies have also highlighted an important role for the transcriptional regulator TOX in driving the epigenetic enforcement of exhaustion, but key questions remain about the potential to reverse the epigenetic programme of exhaustion and how this might affect the persistence of T cell populations.
B cell maturation antigen (BCMA) is a validated target for chimeric antigen receptor (CAR) T cell therapy in multiple myeloma (MM). Despite promising objective response rates, most patients relapse and low levels of BCMA on a subset of tumor cells has been suggested as a probable escape mechanism. BCMA is actively cleaved from the tumor cell surface by the ubiquitous multisubunit γ-secretase complex, which reduces ligand density on tumor cells for CAR-T cell recognition and releases a soluble BCMA (sBCMA) fragment capable of inhibiting CAR-T cell function. Sufficient sBCMA can accumulate in the bone marrow of MM patients to inhibit CAR-T cell recognition of tumor cells, and potentially limit efficacy of BCMA directed adoptive T cell therapy. We investigated whether blocking BCMA cleavage by small molecule γ-secretase inhibitors (GSIs) could augment BCMA-targeted CAR-T cell therapy. We found that exposure of myeloma cell lines and patient tumor samples to GSIs markedly increased surface BCMA levels in a dose-dependent fashion, concurrently decreased sBCMA concentrations, and improved tumor recognition by CAR-T cells in vitro GSI treatment of MM tumor bearing NOD/SCID/γc-/- mice increased BCMA expression on tumor cells, decreased sBCMA in peripheral blood, and improved antitumor efficacy of BCMA-targeted CAR-T cell therapy. Importantly, short term GSI administration to MM patients markedly increases the percentage of BCMA+tumor cells, and the levels of BCMA surface expression in vivo Based on these data, an FDA-approved clinical trial (NCT03502577) has been initiated, combining GSI with concurrent BCMA CAR-T cell therapy.
Despite progress in adoptive T-cell therapies, identification of targets remains a challenge. Although chimeric antigen receptors recognize cell-surface antigens, T-cell receptors (TCRs) have the advantage that they can target the array of intracellular proteins by binding to peptides associated with MHC products (pepMHC). Although hundreds of cancer-associated peptides have been reported, it remains difficult to identify effective TCRs against each pepMHC complex. Conventional approaches require isolation of antigen-specific CD8+ T cells, followed by TCRαβ gene isolation and validation. To bypass this process, we used directed evolution to engineer TCRs with desired peptide specificity. Here, we compared the activity and cross-reactivity of two affinity-matured TCRs (T1 and RD1) with distinct origins. T1-TCR was isolated from a melanoma-reactive T-cell line specific for MART-1/HLA-A2 whereas RD1-TCR was derived de novo against MART-1/HLA-A2 by in vitro engineering. Despite their distinct origins, both TCRs exhibited similar peptide fine-specificities, focused on the center of the MART-1 peptide. In CD4+ T cells, both TCRs mediated activity against MART-1 presented by HLA-A2. However, in CD8+ T cells, T1 but not RD1 demonstrated cross-reactivity with endogenous peptide/HLA-A2 complexes. Based on the fine specificity of these and other MART-1 binding TCRs, we conducted bioinformatics scans to identify structurally similar self-peptides in the human proteome. We showed that the T1-TCR cross-reacted with many of these self-peptides whereas the RD1-TCR was rarely cross-reactive. Thus, TCRs like RD1, generated de novo against cancer antigens, can serve as an alternative to TCRs generated from T-cell clones.
Background:
Ewing sarcoma (EWS) manifests one of the lowest somatic mutation rates of any cancer, leading to a scarcity of druggable mutations and neoantigens. Immunotherapeutics targeting differentially expressed cell surface antigens could provide therapeutic benefit for such tumors. Pregnancy-associated plasma protein A (PAPP-A) is a cell membrane-associated proteinase produced by the placenta that promotes fetal growth by inducing insulinlike growth factor (IGF) signaling.
Methods:
By comparing RNA expression of cell surface proteins in EWS (n = 120) versus normal tissues (n = 42), we comprehensively characterized the surfaceome of EWS to identify highly differentially expressed molecules. Using CRISPR/Cas-9 and anti-PAPP-A antibodies, we investigated biological roles for PAPP-A in EWS in vitro and in vivo in NSG xenograft models and performed RNA-sequencing on PAPPA knockout clones (n = 5) and controls (n = 3). All statistical tests were two-sided.
Results:
EWS surfaceome analysis identified 11 highly differentially overexpressed genes, with PAPPA ranking second in differential expression. In EWS cell lines, genetic knockout of PAPPA and treatment with anti-PAPP-A antibodies revealed an essential survival role by regulating local IGF-1 bioavailability. MAb-mediated PAPPA inhibition diminished EWS growth in orthotopic xenografts (leg area mm2 at day 49 IgG2a control (CTRL) [n = 14], mean = 397.0, SD = 86.1 vs anti-PAPP-A [n = 14], mean = 311.7, SD = 155.0; P = .03; median OS anti-PAPP-A = 52.5 days, 95% CI = 46.0 to 63.0 days vs IgG2a = 45.0 days, 95% CI = 42.0 to 52.0 days; P = .02) and improved the efficacy of anti-IGF-1R treatment (leg area mm2 at day 49 anti-PAPP-A + anti-IGF-1R [n = 15], mean = 217.9, SD = 148.5 vs IgG2a-CTRL; P < .001; median OS anti-PAPP-A + anti-IGF1R = 63.0 days, 95% CI = 52.0 to 67.0 days vs IgG2a-CTRL; P < .001). Unexpectedly, PAPPA knockout in EWS cell lines induced interferon (IFN)-response genes, including proteins associated with antigen processing/presentation. Consistently, gene expression profiles in PAPPA-low EWS tumors were enriched for immune response pathways.
Conclusion:
This work provides a comprehensive characterization of the surfaceome of EWS, credentials PAPP-A as a highly differentially expressed therapeutic target, and discovers a novel link between IGF-1 signaling and immune evasion in cancer, thus implicating shared mechanisms of immune evasion between EWS and the placenta.
Chimeric antigen receptor (CAR) T cell therapy for B cell malignancies has surpassed expectations, driving an ever-expanding number of clinical trials and the first US Food and Drug Administration approvals of cell therapies for the treatment of cancer. This experience has illuminated some generalizable requirements for CAR T cell efficacy as well as the interplay between disease biology and clinical outcomes. Major CAR intrinsic variables affecting T cell behavior have been defined, and mechanisms of tumor resistance are increasingly understood. Here, we review the clinical experience with CAR T cells amassed to date, including but not limited to B cell malignancies, emphasizing factors associated with efficacy, resistance and major barriers to success. We also discuss how these insights are driving next-generation clinical trials, including those in solid tumors. Clinical application of CAR T cells to cancer therapy has met with much success, but future trials must build on current understanding to overcome therapy resistance
Purpose:
Vα24-invariant natural killer T cells (NKTs) are attractive carriers for chimeric antigen receptors (CARs) due to their inherent antitumor properties and preferential localization to tumor sites. However, limited persistence of CAR-NKTs in tumor-bearing mice is associated with tumor recurrence. Here, we evaluated whether co-expression of the NKT homeostatic cytokine IL-15 with a CAR enhances the in vivo persistence and therapeutic efficacy of CAR-NKTs.
Experimental design:
Human primary NKTs were ex vivo expanded and transduced with CAR constructs containing an optimized GD2-specific single-chain variable fragment and either the CD28 or 4-1BB costimulatory endodomain, each with or without IL-15 (GD2.CAR or GD2.CAR.15). Constructs that mediated robust CAR-NKT cell expansion were selected for further functional evaluation in vitro and in xenogeneic mouse models of neuroblastoma (NB).
Results:
Co-expression of IL-15 with either costimulatory domain increased CAR-NKT absolute numbers. However, constructs containing 4-1BB induced excessive activation-induced cell death and reduced numeric expansion of NKTs compared with respective CD28-based constructs. Further evaluation of CD28-based GD2.CAR and GD2.CAR.15 showed that co-expression of IL-15 led to reduced expression levels of exhaustion markers in NKTs and increased multi-round in vitro tumor cell killing. Following transfer into mice bearing NB xenografts, GD2.CAR.15 NKTs demonstrated enhanced in vivo persistence, increased localization to tumor sites, and improved tumor control compared to GD2.CAR NKTs. Importantly, GD2.CAR.15 NKTs did not produce significant toxicity as determined by histopathological analysis.
Conclusions:
Our results informed selection of the CD28-based GD2.CAR.15 construct for clinical testing and led to initiation of a first-in-human CAR-NKT clinical trial (NCT03294954).
Purpose:
T cells engineered to express a chimeric antigen receptor (CAR) against CD19 have recently been FDA approved for the treatment of relapsed or refractory large B-cell lymphoma. Despite the success and curative potential of CD19 CAR T cells, several reports describing disease relapse due to antigen loss are now emerging.
Experimental design:
We developed a novel CAR construct directed against CD79b, a critical receptor for successful B-cell development that remains highly expressed in several subtypes of B-cell lymphoma, including mantle cell lymphoma (MCL). We tested CAR T cells directed against CD79b alone or in combination with CD19 targeting in a single construct, against cell line- and patient-derived xenograft models.
Results:
We demonstrate CAR79b antigen-specific recognition and cytotoxicity against a panel of cell lines and patient-derived xenograft models of MCL. Importantly, we show that downregulation of CD19 does not influence surface expression of CD79b and that anti-CD79b CAR T cells alone or arranged in a dual-targeting format with a CD19 single-chain variable fragment (scFv) are able to recognize and eliminate CD19+, CD19-, and mixed CD19+/CD19- B-cell lymphoma.
Conclusions:
Our findings demonstrate that CAR T cells targeting CD79b alone or in combination have promise for treating and preventing CD19 antigen escape in B-cell lymphomas.
Background:
Anti-B-cell maturation antigen (BCMA) chimeric antigen receptor (CAR) T-cell therapy has been shown to have activity in patients with relapsed or refractory multiple myeloma. Reports have suggested that a small subgroup of less differentiated myeloma clones express CD19 and anti-CD19 CAR T-cell therapy has shown activity in some of these patients. We aimed to assess the activity and safety of a combination of humanised anti-CD19 and anti-BCMA CAR T cells in patients with relapsed or refractory multiple myeloma.
Methods:
We did a single-centre, single-arm, phase 2 trial at the Affiliated Hospital of Xuzhou Medical University in China. Patients were eligible if they were aged 18-69 years, had histologically confirmed multiple myeloma, a Karnofsky Performance Score of 50 points or more, and met the International Myeloma Working Group diagnostic criteria for relapsed or refractory disease. Fludarabine (three daily doses of 30mg/m2) and cyclophosphamide (one daily dose of 750 mg/m2) were used to deplete lymphocytes before infusion of humanised anti-CD19 CAR T cells (1 × 106 cells per kg) and murine anti-BCMA CAR T cells (1 × 106 cells per kg). The primary outcome was the proportion of patients who achieved an overall response. Responses were assessed according to the International Myeloma Working Group criteria. This study is registered with the Chinese Clinical Trial Registration Center, number ChiCTR-OIC-17011272.
Findings:
From May 1, 2017, to Jan 20, 2019, 22 patients were enrolled and 21 received an infusion of CAR T cells and were evaluable for safety and activity analyses. At a median follow-up of 179 days (IQR 72-295), 20 (95%) of 21 patients had an overall response, including nine (43%) stringent complete responses, three (14%) complete responses, five (24%) very good partial responses, and three (14%) partial responses. The most common adverse events included cytokine release syndrome (19 [90%] of 21), including 18 patients (86%) with grade 1-2 cytokine release syndrome. The most common serious adverse events were haematological toxicities, which occurred in 20 (95%) of 21 patients. Common grade 3 or higher adverse events included neutropenia (18 [86%]), anaemia (13 [62%]), and thrombocytopenia (13 [62%]). One patient died due to cerebral hemorrhage, which was considered related to sustained thrombocytopenia. No deaths were judged to be treatment-related.
Interpretation:
Our results confirm that combined infusion of humanised anti-CD19 and anti-BCMA CAR T cells is feasible in patients with relapsed or refractory multiple myeloma, and the preliminary activity observed warrants further investigation in randomised trials. This dual CAR-T cell combinations might be a promising treatment option for relapsed or refractory multiple myeloma.
Funding:
National Natural Science Foundation of China, Natural Science Foundation, Key Research and Development Plan of Jiangsu.
Immunotherapy with chimeric antigen receptor (CAR)–engineered T cells can be effective against advanced malignancies. CAR T cells are “living drugs” that require technologies to enable physicians (and patients) to maintain control over the infused cell product. Here, we demonstrate that the tyrosine kinase inhibitor dasatinib interferes with the lymphocyte-specific protein tyrosine kinase (LCK) and thereby inhibits phosphorylation of CD3ζ and ζ-chain of T cell receptor–associated protein kinase 70 kDa (ZAP70), ablating signaling in CAR constructs containing either CD28_CD3ζ or 4-1BB_CD3ζ activation modules. As a consequence, dasatinib induces a function-off state in CD8 ⁺ and CD4 ⁺ CAR T cells that is of immediate onset and can be sustained for several days without affecting T cell viability. We show that treatment with dasatinib halts cytolytic activity, cytokine production, and proliferation of CAR T cells in vitro and in vivo. The dose of dasatinib can be titrated to achieve partial or complete inhibition of CAR T cell function. Upon discontinuation of dasatinib, the inhibitory effect is rapidly and completely reversed, and CAR T cells resume their antitumor function. The favorable pharmacodynamic attributes of dasatinib can be exploited to steer the activity of CAR T cells in “function-on-off-on” sequences in real time. In a mouse model of cytokine release syndrome (CRS), we demonstrated that a short treatment course of dasatinib, administered early after CAR T cell infusion, protects a proportion of mice from otherwise fatal CRS. Our data introduce dasatinib as a broadly applicable pharmacologic on/off switch for CAR T cells.
Cancer arises from the accumulation of genetic alterations, which can lead to the production of mutant proteins not expressed by normal cells. These mutant proteins can be processed and presented on the cell surface by major histocompatibility complex molecules as neoepitopes, allowing CD8 ⁺ T cells to mount responses against them. For solid tumors, only an average 2% of neoepitopes predicted by algorithms have detectable endogenous antitumor T cell responses. This suggests that low mutation burden tumors, which include many pediatric tumors, are poorly immunogenic. Here, we report that pediatric patients with acute lymphoblastic leukemia (ALL) have tumor-associated neoepitope-specific CD8 ⁺ T cells, responding to 86% of tested neoantigens and recognizing 68% of the tested neoepitopes. These responses include a public neoantigen from the ETV6-RUNX1 fusion that is targeted in seven of nine tested patients. We characterized phenotypic and transcriptional profiles of CD8 ⁺ tumor-infiltrating lymphocytes (TILs) at the single-cell level and found a heterogeneous population that included highly functional effectors. Moreover, we observed immunodominance hierarchies among the CD8 ⁺ TILs restricted to one or two putative neoepitopes. Our results indicate that robust antitumor immune responses are induced in pediatric ALL despite their low mutation burdens and emphasize the importance of immunodominance in shaping cellular immune responses. Furthermore, these data suggest that pediatric cancers may be amenable to immunotherapies aimed at enhancing immune recognition of tumor-specific neoantigens.
Background
Preclinical studies suggest that bb2121, a chimeric antigen receptor (CAR) T-cell therapy that targets B-cell maturation antigen (BCMA), has potential for the treatment of multiple myeloma.
Methods
In this phase 1 study involving patients with relapsed or refractory multiple myeloma, we administered bb2121 as a single infusion at doses of 50×10⁶, 150×10⁶, 450×10⁶, or 800×10⁶ CAR-positive (CAR+) T cells in the dose-escalation phase and 150×10⁶ to 450×10⁶ CAR+ T cells in the expansion phase. Patients had received at least three previous lines of therapy, including a proteasome inhibitor and an immunomodulatory agent, or were refractory to both drug classes. The primary end point was safety.
Results
Results for the first 33 consecutive patients who received a bb2121 infusion are reported. The data-cutoff date was 6.2 months after the last infusion date. Hematologic toxic effects were the most common events of grade 3 or higher, including neutropenia (in 85% of the patients), leukopenia (in 58%), anemia (in 45%), and thrombocytopenia (in 45%). A total of 25 patients (76%) had cytokine release syndrome, which was of grade 1 or 2 in 23 patients (70%) and grade 3 in 2 patients (6%). Neurologic toxic effects occurred in 14 patients (42%) and were of grade 1 or 2 in 13 patients (39%). One patient (3%) had a reversible grade 4 neurologic toxic effect. The objective response rate was 85%, including 15 patients (45%) with complete responses. Six of the 15 patients who had a complete response have had a relapse. The median progression-free survival was 11.8 months (95% confidence interval, 6.2 to 17.8). All 16 patients who had a response (partial response or better) and who could be evaluated for minimal residual disease (MRD) had MRD-negative status (≤10⁻⁴ nucleated cells). CAR T-cell expansion was associated with responses, and CAR T cells persisted up to 1 year after the infusion.
Conclusions
We report the initial toxicity profile of a BCMA-directed cellular immunotherapy for patients with relapsed or refractory multiple myeloma. Antitumor activity was documented. (Funded by Bluebird Bio and Celgene; CRB-401 ClinicalTrials.gov number, NCT02658929.)
QUICK TAKE VIDEO SUMMARY
A Novel CAR T-Cell Therapy for Multiple Myeloma
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Chimeric antigen receptor (CAR) T cell therapies have achieved promising outcomes in several cancers, however more challenging oncology indications may necessitate advanced antigen receptor designs and functions. Here we describe a bipartite receptor system comprised of separate antigen targeting and signal transduction polypeptides, each containing an extracellular dimerization domain. We demonstrate that T cell activation remains antigen dependent but can only be achieved in the presence of a dimerizing drug, rapamycin. Studies performed in vitro and in xenograft mouse models illustrate equivalent to superior anti-tumor potency compared to currently used CAR designs, and at rapamycin concentrations well below immunosuppressive levels. We further show that the extracellular positioning of the dimerization domains enables the administration of recombinant re-targeting modules, potentially extending antigen targeting. Overall, this novel regulatable CAR design has exquisite drug sensitivity, provides robust anti-tumor responses, and is uniquely flexible for multiplex antigen targeting or retargeting, which may further assist the development of safe, potent and durable T cell therapeutics.
Background:
Chimeric antigen receptor (CAR) T cells are a promising therapy for hematologic malignancies. B-cell maturation antigen (BCMA) is a rational target in multiple myeloma (MM).
Methods:
We conducted a phase I study of autologous T cells lentivirally-transduced with a fully-human, BCMA-specific CAR containing CD3ζ and 4-1BB signaling domains (CART-BCMA), in subjects with relapsed/refractory MM. Twenty-five subjects were treated in 3 cohorts: 1) 1-5 x 108 CART-BCMA cells alone; 2) Cyclophosphamide (Cy) 1.5 g/m2 + 1-5 x 107 CART-BCMA cells; and 3) Cy 1.5 g/m2 + 1-5 x 108 CART-BCMA cells. No pre-specified BCMA expression level was required.
Results:
CART-BCMA cells were manufactured and expanded in all subjects. Toxicities included cytokine release syndrome and neurotoxicity, which were grade 3-4 in 8 (32%) and 3 (12%) subjects, respectively, and reversible. One subject died at day 24 from candidemia and progressive myeloma, following treatment for severe CRS and encephalopathy. Responses (based on treated subjects) were seen in 4/9 (44%) in cohort 1, 1/5 (20%) in cohort 2, and 7/11 (64%) in cohort 3, including 5 partial, 5 very good partial, and 2 complete responses, 3 of which were ongoing at 11, 14, and 32 months. Decreased BCMA expression on residual MM cells was noted in responders; expression increased at progression in most. Responses and CART-BCMA expansion were associated with CD4:CD8 T cell ratio and frequency of CD45RO-CD27+CD8+ T cells in the pre-manufacturing leukapheresis product.
Conclusion:
CART-BCMA infusions with or without lymphodepleting chemotherapy are clinically active in heavily-pretreated MM patients.
Trial registration:
NCT02546167.
Funding:
University of Pennsylvania-Novartis Alliance and NIH.
Regulatory T cells (Tregs) are key modulators of inflammation and are important for the maintenance of peripheral tolerance. Adoptive immunotherapy with polyclonal Tregs holds promise in organ transplantation, graft-versus-host disease, and autoimmune diseases, but may be enhanced by antigen-specific, long-lived Treg cells. We modified primary human Tregs with chimeric antigen-receptors (CARs) bearing different costimulatory domains and performed in vitro analyses of their phenotype and function. While neither the presence of a CAR nor the type of costimulation domain influenced Foxp3 expression in Tregs, the costimulation domain of the CARs affected CAR Treg surface phenotype and functions such as cytokine production. Furthermore, signaling from the CD28 costimulation domain maintained CAR Treg suppressor function, whereas 4-1B costimulation did not. In vivo, CAR Tregs accumulated at sites expressing target antigen, and suppressed antigen specific effector T cell responses; however, only CAR Tregs with CD28 signaling domains were potent inhibitors of effector T cell mediated graft rejection in vivo. Our findings support the use of CD28 based CAR-Tregs for tissue specific immune suppression in the clinic.
Key Points
Dasatinib potently and reversibly suppresses CAR-T cell cytotoxicity, cytokine secretion, and proliferation. Dasatinib could be repurposed as a safety switch to mitigate CAR-mediated toxicity in patients.
Adoptive cell therapy (ACT) with T cells targeting neoantigens can mediate durable responses in patients with metastatic cancer. Cell therapies targeting common shared antigens for epithelial cancers are not yet broadly available. Here we report the identification and characterization in one patient of T cell receptors (TCRs) recognizing mutated p53 p.R175H, which is shared among a subset of patients with cancer. Tumor-infiltrating lymphocytes (TILs) were screened for recognition of mutated neoantigens in a patient with metastatic colorectal cancer. HLA-A*0201 restricted recognition of mutated p53 p.R175H was identified, and the minimal peptide epitope was HMTEVVRHC. Reactive T cells were isolated by tetramer sorting, and three TCRs were identified. These TCRs mediated recognition of commercially available ovarian cancer, uterine carcinoma, and myeloma cell lines, as well as an NIH patient-derived esophageal adenocarcinoma line that endogenously expressed p53 p.R175H and HLA-A*0201. They also mediated recognition of p53 p.R175H+ colon, breast, and leukemia cell lines after transduction with a retrovirus encoding HLA-A*0201. This work demonstrates that common shared mutated epitopes like those found in p53 can elicit immunogenic responses and that the application of ACT may be extended to patients with any cancer histology that expresses both HLA-A*0201 and the p53 p.R175H mutation.
Autologous T cells targeting Epstein Barr virus (EBV) latent membrane proteins (LMPs) have shown safety and efficacy for the treatment of patients with type II latency EBV-associated lymphomas who have failed standard therapies, including high dose chemotherapy followed by autologous stem cell rescue. However, the safety and efficacy of allogeneic donor-derived LMP-specific T cells (LMP-Ts) have not been established for patients who have received allogeneic HSCT. Thus, we evaluated the safety and efficacy of donor-derived LMP-Ts in 26 patients who had received an allogeneic hematopoietic stem cell transplant (HSCT) for EBV-associated T/NK or B cell lymphomas (NCT00062868, NCT01956084; ClinicalTrials.gov). Seven patients received LMP-Ts as therapy for active disease, and 19 were treated as adjuvant therapy for high-risk disease. There were no immediate infusion-related toxicities and only one dose-limiting toxicity potentially related to T cell infusion. The two-year overall survival (OS) was 68%. Additionally, patients who received T cell therapy while in complete remission after allogeneic HSCT had a 78% OS at 2 years. Patients treated for B cell diseases (n=10) had a 2-year OS of 80%. Patients with T cell diseases had a 2 year OS of 60% which suggests an improvement compared to published post-transplant 2-year overall survival rates of 30-50%.1 Hence, this study shows that donor-derived LMP-Ts are a safe and effective therapy to prevent relapse after transplant in patients with B or T cell-derived EBV-associated lymphomas or lymphoproliferative disorders and supports the infusion of LMP-specific T cells as adjuvant therapy to improve outcomes in the post-transplant setting.
Emerging data from chimeric antigen receptor (CAR) T-cell trials in B-cell malignancies demonstrate that a common mechanism of resistance to this novel class of therapeutics is the emergence of tumors with loss or downregulation of the target antigen. Antigen loss or antigen-low escape is likely to emerge as an even greater barrier to success in solid tumors, which manifest greater heterogeneity in target antigen expression. Potential approaches to overcome this challenge include engineering CAR T cells to achieve multispecificity and to respond to lower levels of target antigen and more efficient induction of natural antitumor immune responses as a result of CAR-induced inflammation. In this article, we review the evidence to date for antigen escape and downregulation and discuss approaches currently under study to overcome these obstacles.
Significance: Antigen escape and downregulation have emerged as major issues impacting the durability of CAR T-cell therapy. Here, we explore their incidence and ways to overcome these obstacles in order to improve clinical outcomes.
Chimeric antigen receptors (CARs) have shown remarkable ability to re-direct T cells to target CD19-expressing tumours, resulting in remission rates of up to 90% in individuals with paediatric acute lymphoblastic lymphoma. Lessons learned from these clinical trials of adoptive T cell therapy for cancer, as well as investments made in manufacturing T cells at commercial scale, have inspired researchers to develop CARs for additional applications. Here, we explore the challenges and opportunities of using this technology to target infectious diseases such as with HIV and undesired immune responses such as autoimmunity and transplant rejection. Despite substantial obstacles, the potential of CAR T cells to enable cures for a wide array of disease settings could be transformational for the medical field.
Chimeric antigen receptors (CARs) significantly enhance the anti-tumor activity of immune effector cells. Although most studies have evaluated CAR expression in T cells, here we evaluate different CAR constructs that improve natural killer (NK) cell-mediated killing. We identified a CAR containing the transmembrane domain of NKG2D, the 2B4 co-stimulatory domain, and the CD3ζ signaling domain to mediate strong antigen-specific NK cell signaling. NK cells derived from human iPSCs that express this CAR (NK-CAR-iPSC-NK cells) have a typical NK cell phenotype and demonstrate improved anti-tumor activity compared with T-CAR-expressing iPSC-derived NK cells (T-CAR-iPSC-NK cells) and non-CAR-expressing cells. In an ovarian cancer xenograft model, NK-CAR-iPSC-NK cells significantly inhibited tumor growth and prolonged survival compared with PB-NK cells, iPSC-NK cells, or T-CAR-iPSC-NK cells. Additionally, NK-CAR-iPSC-NK cells demonstrate in vivo activity similar to that of T-CAR-expressing T cells, although with less toxicity. These NK-CAR-iPSC-NK cells now provide standardized, targeted “off-the-shelf” lymphocytes for anti-cancer immunotherapy.
We evaluated the safety and activity of autologous T cells expressing NY-ESO-1c259, an affinity-enhanced T-cell receptor (TCR) recognizing an HLA-A2–restricted NY-ESO-1/LAGE1a–derived peptide, in patients with metastatic synovial sarcoma (NY-ESO-1c259T cells). Confirmed antitumor responses occurred in 50% of patients (6/12) and were characterized by tumor shrinkage over several months. Circulating NY-ESO-1c259T cells were present postinfusion in all patients and persisted for at least 6 months in all responders. Most of the infused NY-ESO-1c259T cells exhibited an effector memory phenotype following ex vivo expansion, but the persisting pools comprised largely central memory and stem-cell memory subsets, which remained polyfunctional and showed no evidence of T-cell exhaustion despite persistent tumor burdens. Next-generation sequencing of endogenous TCRs in CD8+ NY-ESO-1c259T cells revealed clonal diversity without contraction over time. These data suggest that regenerative pools of NY-ESO-1c259T cells produced a continuing supply of effector cells to mediate sustained, clinically meaningful antitumor effects.
Significance: Metastatic synovial sarcoma is incurable with standard therapy. We employed engineered T cells targeting NY-ESO-1, and the data suggest that robust, self-regenerating pools of CD8+ NY-ESO-1c259T cells produce a continuing supply of effector cells over several months that mediate clinically meaningful antitumor effects despite prolonged exposure to antigen. Cancer Discov; 8(8); 944–57. ©2018 AACR.
See related commentary by Keung and Tawbi, p. 914.
This article is highlighted in the In This Issue feature, p. 899