T-cell therapies have transformed the treatment landscape for hematologic malignancies and show growing promise for solid tumors and non-oncologic diseases. Unlike conventional drugs, these living therapeutics are shaped not only by genetic engineering but also by the ex vivo manufacturing process itself, which functions as a powerful biological determinant of cell fate and function. Here, we present a framework that conceptualizes manufacturing as a form of biological programming that governs therapeutic outcomes through its effects on T-cell phenotype, fitness, and functional durability. We review how each major processing step, including isolation, selection, activation, transduction, expansion, and formulation, influences the balance between cell quantity and quality, the two principal determinants of clinical efficacy. Particular emphasis is placed on how extracellular cues encountered during manufacturing, such as physical properties of activation materials, stimulatory ligand presentation, cytokine and nutrient composition, and gene delivery method, shape transcriptional, epigenetic, and metabolic programs that define cell phenotypes directly linked to cell quantity and quality. Emerging strategies to deliberately steer T-cell differentiation toward persistence-associated phenotypes, enhance metabolic fitness, and reduce vein-to-vein time are highlighted as critical avenues for next-generation manufacturing. Additionally, reducing inconsistency and cost remain areas of strategic importance to expand availability. Collectively, this review underscores that optimizing T-cell therapy requires treating manufacturing not as a logistical necessity but as a controllable biological intervention. A mechanistic understanding of how processing decisions program cellular behavior will enable the rational design of more potent, durable, and scalable T-cell products for diverse clinical applications.
T cells endowed with defined antigen specificity by provision of monoclonal T cell receptors (TCR) or synthetic Chimeric Antigen Receptors (CAR) have established themselves as a potent drug class with several such products now commercially available. This review addresses fundamental aspects of the design of engineered TCR and CAR T therapies. Beyond the basic design features, we touch upon several additional strategies that have been developed or are currently in pre-clinical development to enhance the efficacy or safety of such therapies. Within the vast and quickly growing space of T cell engineering, this discussion is not all-encompassing but aims to lay a foundation for other reviews in the series that delve into more focused aspects of T cell therapy.
Engineered cell therapies have shown remarkable promise in treating malignancies and autoimmune diseases. As of 2025, there are seven FDA-approved chimeric antigen receptor (CAR) T therapies, each treating different cancers. Despite rapid progress in developing new therapies, there is a lack of understanding about the in vivo behavior of adoptively transferred cells. Noninvasive methods that monitor CAR T cell dynamics and persistence in vivo are critical to both assess individual patients' responses to therapy in real time and to guiding improvements to engineered cell therapies. Imaging approaches that evaluate persistence, proliferation, functionality, and distribution of T cells will aid in optimizing therapeutic development and adjustment of treatment strategies in the clinic. Molecular imaging can noninvasively track labeled cells on a whole-body level and enable long-term monitoring of adoptively transferred cells in a manner compatible with the proliferation and persistence of therapeutic cells and may someday be able to bypass the need for paired biopsies. In this chapter, we highlight examples of cell tracking and labeling strategies for engineered T cell therapies.
Chimeric antigen receptor (CAR) T cell therapy has achieved clinical success in hematological malignancies, but its reliance on viral vectors and complex ex vivo manufacturing poses challenges related to safety, cost, and scalability. Next-generation strategies, including universal ("off-the-shelf") and in vivo CAR-T cell therapies, have emerged to address these limitations. The latter strategy employs targeted delivery systems to directly program patients' T cells in situ, bypassing ex vivo manipulation and offering a more streamlined, scalable, and safer therapeutic paradigm. The success of in vivo CAR-T cell therapy relies on targeted delivery systems. While engineered lentiviruses enable stable integration, non-viral vectors for transient CAR expression offer superior pharmacological control. This approach, exemplified by lipid nanoparticles in combination with mRNA, avoids risks of insertional mutagenesis and enables titratable, short-lived CAR expression, thereby enhancing safety management and suitability for applications beyond oncology. In this chapter, we first delineate the pharmacological imperative for transient CAR expression. Next, various delivery strategies are systematically reviewed, including ex vivo electroporation, in vivo non-viral systems, and engineered virus-like particles. Afterwards, we summarize the ongoing clinical trials of transient CAR-T cell therapy for oncology and non-oncology indications. Finally, we provide perspectives on the development of next-generation transient CAR-T cell therapies.
Therapeutic T cells show enormous promise for the treatment of cancer and other diseases, and an intriguing attribute of T cells for cellular therapy is their ability to have persist and perform cytotoxic function for years. With the advances in T-cell therapies such as CAR T-cell therapy for cancer, long-persisting therapeutic T cells have now been studied in clinical settings and revealed unexpected T-cell poulations. Based on these studies, an emerging framework of long-lived cytotoxic T cells may guide the rational design of new therapies optimized for long-lasting efficacy. The ideal duration of therapeutic T-cell persistence varies by clinical context, and the design of T-cell therapies should follow the therapeutic objective. This chapter highlights the central objective of enhancing the persistence of therapeutic T cells in various clinical contexts, and discusses the ways in which our growing knowledge of long-persisting T cells can guide the next generation of cell therapies.
Unconventional T cells, including gamma delta (γδ) T cells, mucosal-associated invariant T (MAIT) cells, and CD1d-restricted natural killer T (NKT) cells, comprise a unique component of the immune system. These cells play critical roles in host defense, immune regulation, and disease pathogenesis. In particular, invariant NKT (iNKT) cells have emerged as key immunological orchestrators that bridge innate and adaptive immunity by rapidly recognizing lipid antigens presented by CD1d molecules. This chapter provides a comprehensive overview of iNKT cell biology, including their development, phenotypic and functional heterogeneity, and activation mechanisms. It discusses the role of iNKT cells in pathological conditions in detail. Their ability to directly kill tumor cells, as well as their potential to orchestrate the immune response, opens up new possibilities for anti-cancer therapy. Their contribution to immune response regulation and tolerance highlights their critical role in infectious diseases and transplantation. The chapter summarizes current clinical approaches aimed at harnessing iNKT cells, including in vivo activation strategies, adoptive transfer of ex vivo-expanded cells, and developing chimeric antigen receptor (CAR)-engineered iNKT cells. These emerging therapies have advantages over conventional CAR-T approaches, such as reduced toxicity and the potential for allogeneic use. Finally, the chapter discusses the role of type II NKT cells and their cross-regulatory interactions with iNKT cells. Overall, CD1d-restricted NKT cells are a promising target for next-generation immunotherapies. However, further mechanistic and clinical studies are needed to realize their full therapeutic potential.
Adoptive cell therapies, particularly chimeric antigen receptor (CAR) T cells, function as "living drugs" whose efficacy depends not only on target recognition but also on the metabolic state of the infused product. T cell metabolism governs energy production, redox homeostasis, biomass generation, and adaptation to persistent antigen exposure and nutrient stress, thereby shaping expansion, effector function, persistence, and susceptibility to exhaustion. Core metabolic programs relevant to these outcomes include glycolysis and mitochondrial respiration, anaplerosis and amino acid metabolism, lipid metabolism, and NAD- and redox-linked pathways. These programs help determine adoptive cell therapy-relevant phenotypes, including the balance between immediate cytotoxicity and long-term durability. Increasing evidence further suggests that metabolism can be therapeutically manipulated across the lifecycle of adoptive cell therapy through ex vivo manufacturing, receptor and signaling design, direct gene engineering, and post-infusion support. Collectively, these findings support a pharmacologic framework in which metabolic state is not merely a descriptive correlate of product quality, but a controllable determinant of therapeutic performance. A deeper mechanistic understanding of these pathways may enable more precise strategies to improve persistence, function, and long-term antitumor efficacy.
Therapeutic proteins have emerged as potential candidates for the management of neurodegenerative disorders affecting the central nervous system (CNS). However, their effective delivery to the CNS remains a major challenge, primarily due to the presence of the blood-brain barrier (BBB). To overcome this challenge, several brain-targeted delivery strategies based on peptides have been developed. These peptides harness endogenous BBB transport pathways to enhance the delivery of therapeutic proteins to the CNS. In order to develop new potential protein-based therapeutic candidates, considerable efforts have focused on engineering fusion proteins that combine therapeutic efficacy with enhanced BBB permeability.
Lipid nanoparticles (LNPs) have gained growing attention as advanced drug delivery systems capable of addressing major challenges in the development of innovative therapies. They have been widely investigated to overcome the challenges of development delivery strategies for the Central Nervous System (CNS). Their small size, biomimetic lipid composition, and modifiable surface properties make them promising candidates for targeting the Blood Brain Barrier (BBB), leading to improved therapeutic interventions. This chapter provides an integrative overview of the main classes of LNPs and their manufacturing methods, offering insights into their design, characterization, and optimization for brain delivery. We first discuss the key physicochemical parameters -particle size, polydispersity index, surface charge, shape, crystallinity, and encapsulation efficiency- and their impact on biodistribution, stability, and BBB penetration. Formulation and manufacturing strategies are examined, from conventional approaches to microfluidic mixing. Special attention is given to surface modification strategies, including ligand conjugation and the technological variables influencing receptor-specific transport that determine brain targeting. We place particular emphasis on brain-shuttle peptides, which constitute the most widely employed approach for functionalizing LNPs. Finally, we summarize representative preclinical studies, emerging clinical trials, and key translational challenges -such as neurotoxicity, batch-to-batch reproducibility, and regulatory hurdles-, but also highlight the proved potential of LNPs to achieve successful clinical translation in other therapeutic areas. These advances provide valuable insights and technical precedents that may benefit the development of LNP-based therapeutics for CNS disorders, supporting future strategies targeting neurological and psychiatric disorders as well as brain tumors.
