Tracing the development of CAR-T cell design: from concept to next-generation platforms

Abstract

Chimeric Antigen Receptor (CAR)-T cell therapy represents a transformative breakthrough in cancer immunotherapy by harnessing the adaptive immune system to selectively eradicate cancer cells. Pioneering advances in the treatment of hematological malignancies have led to the FDA approval of several CAR-T cell therapies, particularly for patients with relapsed or refractory disease. This success is a result of continuous refinements in CAR architecture, which have evolved from early prototypes with limited therapeutic efficacy to advanced next-generation receptors that incorporate co-stimulatory domains, cytokine signaling, safety switches, and precision control mechanisms. This review elucidates the fundamental rationale behind CAR-T cell development and addresses key biological challenges encountered. Advances in receptor engineering, metabolic reprogramming, and optimized immune signaling have markedly enhanced the persistence, antitumor activity, and safety profiles of CAR-T cells. Additionally, emerging genetic engineering tools, including CRISPR, base editing, prime editing, and RNA and epigenome editing, hold promise for reducing immunogenicity and minimizing the risk of graft-versus-host disease (GVHD). However, CAR-T cell therapy continues to face several challenges, including severe side effects such as cytokine release syndrome (CRS) and neurotoxicity, inconsistent therapeutic responses, and high production costs. To overcome these barriers, novel approaches are under development that include generating CAR-T cells in vivo, utilizing logic-gated CAR systems, and expanding CAR platforms to include other immune effector cells, such as natural killer cells (CAR-NK) and macrophages (CAR-M). The future of CAR-based therapies is expected to integrate synthetic biology, immune checkpoint modulation, and innovative delivery methods to enhance both therapeutic efficacy and safety. This review synthesizes current knowledge and emerging strategies to guide future advancements aimed at expanding the applicability of CAR therapy to various cancer types and potentially other diseases.

Keywords: CAR design; CAR-T cell therapy; T cell engineering; adoptive cell therapy; gene editing; immunotherapy; next-generation CAR; synthetic immunology.

Figure 1

Timeline of CAR-based immunotherapies. The figure illustrates the historical development of CAR-based therapies, highlighting major milestones from the foundational era to recent advancements in next-generation CAR engineering. Created with BioRender.

Figure 2

Structures of different generations of CAR. The figure depicts the structure of the five generations of CAR-T cells. (A) Early-generation CARs. (B) Next-generation CARs. Created with BioRender.

Figure 3

Types of spacer domains in CAR design. The figure illustrates various types of spacer (hinge) regions that link the antigen-binding domain (scFv) to the transmembrane domain in CARs. IgG-based spacers include Fc regions containing CH2-CH3 domains, Fc regions modified by mutation or deletion to prevent FcγR binding, and truncated variants lacking the CH2-CH3 domains (left). Non-IgG-based spacers are derived from the extracellular stalks of natural T cell proteins, such as CD8α and CD28 hinge regions, or incorporate the nerve growth factor receptor (NGFR), which can function both as a structural spacer and as a therapeutic or selection/tracking marker (right). Created with BioRender.

Figure 4

Boolean logic-gate strategies in CAR-T cells. The figure illustrates four types of logic-gated CAR-T cell circuits designed to enhance tumor specificity and reduce off-target effects: (A) AND-gate: CAR-T cells are activated only when both antigens A and B are present, (B) OR-gate: CAR-T cells are activated when either antigen A or B is present, (C) NOT-gate: CAR-T cell activation is inhibited by the presence of antigen B; and D: AND-NOT gate: CAR-T cell activation requires the presence of both antigens A and B, but the absence of antigen (C) Created with BioRender.

Figure 5

Functional classification of CAR engineering strategies. The figure categorizes different CAR engineering strategies based on their functional objectives, including enhancing specificity and effector function, enhancing safety and reducing toxicity, improving persistence and activation, overcoming tumor microenvironment (TME) suppression, and addressing manufacturing and logistical challenges. For each category, a representative example is visually depicted to illustrate the underlying concept. Created with BioRender.