CAR工程解决方案

Chimeric antigen receptors (CARs) are synthetic, engineered receptors that enable immune cells to recognize, bind to, and eliminate cells expressing a specific target antigen. As this binding is independent of major histocompatibility complex (MHC) presentation, it can be used to target a wide range of antigens and is limited only by antibody availability. CAR-based therapeutics have transformed the field of immunotherapy by allowing precise control over immune cell specificity and activation.

A typical CAR construct comprises four main components (Figure 5A): (i) an extracellular antigen-recognition domain (generally a single-chain variable fragment, or scFv), (ii) an extracellular hinge region that provides flexibility and connects the antigen-recognition domain to the transmembrane domain, (iii) a transmembrane domain that anchors the CAR to the host cell membrane, and (iv) intracellular signaling domain(s). Upon binding to the target antigen on the target cell (Figure 5B), the CAR transmits signals into the engineered immune cell, triggering downstream signaling pathways that drive cell activation, cytokine release, proliferation, and cytotoxic activity. Second- and third-generation CAR constructs incorporate one or more co-stimulatory domains (such as CD28 or 4-1BB) to enhance persistence, potency, and long-term efficacy.

CAR T-cell therapy, the most clinically advanced CAR modality, involves engineering patient- or donor-derived T cells to express CARs that recognize cancer antigens. Upon infusion into patients, CAR T-cells can expand in vivo and mediate potent, antigen-specific tumor cell killing. CAR-T therapies have demonstrated remarkable clinical success in hematologic malignancies, leading to multiple regulatory approvals. However, challenges such as cytokine release syndrome, neurotoxicity, antigen escape, and complex manufacturing have driven continued innovation in CAR design and delivery.

Beyond T cells, CAR technology has been applied to engineer other immune cell types, substantially expanding therapeutic possibilities. CAR–natural killer (NK) cells leverage the innate cytotoxic properties of NK cells and offer an enhanced safety profile compared with CAR T-cells, owing to mitigation of cytokine release syndrome and a reduced risk of graft-versus-host disease. In addition, CAR-NK cells can be manufactured in advance from healthy donors, enabling “off-the-shelf” drug products and prompt patient treatment.

Similarly, CAR-macrophages (CAR-M) demonstrate a favorable safety profile, with early clinical trials reporting no severe cytokine release syndrome, as well as “off-the-shelf” potential. By harnessing the phagocytic and antigen-presenting functions of macrophages, CAR-M therapies exhibit improved infiltration into solid tumors, remodeling of the tumor microenvironment, and enhanced activation of endogenous immune responses. Although these platforms have been most extensively studied in oncology, their potential applications in autoimmune diseases, infectious diseases, and neuroinflammatory disorders are also under active investigation.

Selecting the optimal CAR vector type is a critical design decision that directly influences the safety, efficacy, manufacturability, and clinical applicability of the final drug product. Functional performance can be aligned with specific therapeutic goals by considering the following factors early in the drug discovery pipeline:

• Intended application
• Target cell type
• Desired CAR expression duration
• Dosing strategy
• Tolerance for genomic integration risk

For applications such as autologous (derived from a patient’s own body) and allogeneic (derived from a donor) CAR therapies where stable, long-term CAR expression is required, viral vector systems are generally preferred. These vectors enable permanent genomic integration, supporting sustained CAR expression and in vivo persistence of engineered cells. Lentiviral-based CAR therapies, in particular, have been extensively studied and are a well-established treatment modality across multiple hematologic malignancies. More recently, lentiviral-based in vivo CAR delivery has emerged as an alternative to ex vivo manufacturing, enabling direct transduction of endogenous immune cells within the patient to simplify manufacturing, accelerate treatment initiation, and broaden patient eligibility. Together, these advances highlight the evolving versatility of lentiviral CAR platforms and their potential to further expand the reach and impact of CAR-based therapies across diverse clinical indications.

Conversely, non-viral vector approaches represent attractive alternatives when transient or controllable CAR expression is desired. Delivery via lipid nanoparticle (LNP) encapsulated in vitro transcribed (IVT) RNA enables rapid, non-integrating CAR expression. Its efficient and targeted CAR delivery as well as reduced immunogenicity profile also makes it particularly appealing for therapeutic development. Other non-viral systems, such as plasmid DNA or transposon-based platforms, offer additional flexibility (especially with respect to cargo capacity) and lower production costs. However, gene delivery typically relies on electroporation and requires careful optimization to balance transfection efficiency with cell viability. Nonetheless, these platforms can be tailored to achieve CAR expression profiles suited to their intended therapeutic applications.

Ultimately, CAR vector selection involves careful consideration of expression stability, resulting safety profile, and manufacturing feasibility. As vector technologies and engineering strategies continue to advance, aligning these tools with therapeutic objectives will be critical to maximizing the performance, scalability, and clinical impact of next-generation CAR-based therapies.

Efficiency and efficacy of CAR constructs can be greatly enhanced through careful optimization of the vector backbone and CAR components as well as consideration of the target cell type and therapeutic application. Key strategies include:

• Viral pseudotyping
• Incorporation of CRISPR/Cas genome editing
• CAR construct design

Viral pseudotyping can significantly improve transduction efficiency and targeting specificity. This is particularly relevant for emerging in vivo CAR-T approaches, whereby optimized enveloped pseudotypes can improve selective immune cell uptake, increase in vivo gene transfer efficiency, and support more consistent and durable CAR expression. While lentiviral vectors pseudotyped with BaEV were previously reported to improve transduction of human T cell and NK cells, their poor manufacturing performance due to high toxicity towards producer cells and production variability is a significant limitation. VSV-G engineering is therefore the most widely used and established pseudotyping strategy for viral vector systems. Further refinement of VSV-G expression and functionality can support scalable production, higher in vivo transduction efficiency, more consistent CAR expression, and improved feasibility of systemic, repeat-dosable CAR-T administration.

CRISPR/Cas genome editing enables targeted knockouts of inhibitory receptors (e.g. PD-1) or other regulators of immune inactivation, resulting in increased CAR signaling and persistence. Additionally, CRISPR can also be used to ensure precise integration of the CAR transgene into safe harbor loci, such as the TRAC locus in T cells, promoting uniform expression, reducing insertional mutagenesis risk, and supporting consistent functionality. Collectively, these strategies allow for optimization of cell-intrinsic signaling and receptor expression, leading to enhanced in vivo expression and cytotoxicity.

Rationally, design of the CAR construct itself also plays a large role in therapeutic efficacy. Optimizations of the antigen-recognition, hinge, transmembrane, and intracellular signaling domains can modulate CAR affinity, robustness, and expression stability. For example, the use of dual co-stimulatory domains has been demonstrated to improve both rapid initial activation and long-term survival. Additionally, improved or novel CAR designs through antibody discovery can further enhance CAR activity. For instance, the use of armored CARs or inducible CARs has been shown to improve anti-tumor activity and modulate the tumor microenvironment through secretion of additional cytokines or immune modulators in response to antigen engagement. Beyond CAR architecture, promoter choice also strongly influences the level, duration, and cell-type specificity of CAR expression. While constitutive promoters enable robust expression across transduced cells, cell-specific promoters can confine CAR expression to defined immune subsets for limited off-target effects and improved safety. In emerging in vivo CAR-T approaches, promoter engineering is especially vital as it enables efficient CAR expression while restricting it to functional immune effector cells.

In practice, a combination of these approaches are employed to develop next-generation CAR constructs with superior transduction efficiency, cytotoxicity, and long-term functional persistence. An integrated strategy allows development of therapies that are not only more effective against their target cells, but are also safer, more predictable, and adaptable to diverse CAR-based platforms.