2025.08.16
Chimeric Antigen Receptor T cell (CAR-T) therapy, a groundbreaking immunotherapy, has demonstrated remarkable efficacy in treating hematological cancers such as B-cell lymphoma[1]. While traditional CAR-T therapy relies on an "ex-vivo engineering" model, the emerging "in-vivo engineering" (in-vivo CAR-T) technology aims to generate CAR-T cells directly within the patient’s body, addressing critical bottlenecks of the ex-vivo approach. Based on relevant literature, this article focuses on the technical principles of in-vivo CAR-T, detailing its differences from traditional ex-vivo CAR-T, advantages, challenges, and future prospects, providing a comprehensive analysis of advancements and hurdles in this emerging field.
I. Technical Principles: Mechanisms of In-Vivo CAR-T
The core of in-vivo CAR-T technology lies in directly modifying T cells within the patient to express chimeric antigen receptors (CARs), enabling targeted cancer cell elimination. This is achieved via engineered vector delivery, eliminating the need for ex-vivo isolation and modification steps[2][3]. Its mechanisms involve the following key steps:
Vector Delivery and T Cell Targeting: Non-viral or viral-based vectors are used to deliver CAR genes directly into the patient. These vectors include lipid-based nanoparticles (LNPs)[4], fusogenic nanovesicles (FuNVs)[5], engineered exosomes[6][7], or injectable viral vectors (e.g., lentiviruses)[8][9]. Designed to specifically target circulating T cells, these vectors deliver CAR-encoding genetic material into T cells via membrane fusion or endocytosis[5][10]. For example, lipid nanoparticles can stably deliver CAR genes in vivo, inducing CAR expression in T cells within days to form functional CAR-T cells[10].
Genetic Engineering and CAR Expression: The delivered genetic material (e.g., mRNA or DNA) is expressed as CAR proteins in T cells. CARs typically consist of an antigen-binding domain (e.g., scFv targeting CD19 or BCMA), a transmembrane domain, and intracellular signaling domains[11][1]. In vivo, this process relies on vector optimization to maximize transduction efficiency. For instance, non-viral vectors reduce virus-related safety risks while achieving high transduction efficiency[12][4]. Studies have shown that specific vector delivery systems can induce sustained CAR expression in vivo, enhancing T cell anti-tumor activity[9][5].
Immune Activation and Tumor Killing: In-vivo generated CAR-T cells recognize cancer cell surface antigens, activate T cell signaling pathways, and release cytokines (e.g., IFNγ, TNF) and granzymes to directly eliminate tumor cells[13][10]. Additionally, the system can be designed as a "universal platform" (e.g., combined with immunomodulators like selinexor) to optimize T cell function and reduce microenvironmental suppression[13][14].
The principle of in-vivo CAR-T is essentially "in-situ engineering," simplifying processes to enable real-time immune reprogramming[3][4]. Current technologies are primarily validated in mouse models, with success dependent on vector design ensuring targeting accuracy and gene expression stability[8][9]. This approach avoids cumbersome ex-vivo manufacturing steps, generating CAR-T cells directly within the biological environment[2][8].
II. Differences from Traditional Ex-Vivo CAR-T
Ex-vivo CAR-T, the current clinical mainstream, involves collecting T cells from patients or donors, engineering them via viral transduction or electroporation in laboratories, expanding them, and infusing them back into patients[1][1][15]. The in-vivo model differs significantly in the following aspects:
Manufacturing Process:
1. Ex-vivo CAR-T: Requires multiple ex-vivo steps: leukapheresis, viral transduction (e.g., lentivirus), ex-vivo activation and expansion (2–3 weeks), and final infusion[16][8][15]. This process demands strict laboratory control and carries risks of T cell exhaustion and functional loss[16][17].
2. In-vivo CAR-T: Eliminates all ex-vivo steps; patients receive direct vector injection, with in-vivo systems automatically completing T cell targeting, genetic engineering, and CAR expression[3][9][5]. This drastically simplifies the process, reducing treatment time to days[10].
Cost and Accessibility:
1. Ex-vivo CAR-T: High manufacturing costs (hundreds of thousands of dollars) and reliance on specialized centers (e.g., cell therapy units) limit global accessibility[18][8][19]. For example, FDA-approved CAR-T therapies face manufacturing bottlenecks, restricting use to specific patient groups[18][1].
2. In-vivo CAR-T: Reduces production costs due to simpler vector manufacturing compared to cell culture; can be administered in general medical facilities without specialized centers[18][20]. Studies indicate potential for widespread adoption, particularly in resource-limited regions[2][15].
Efficacy and Toxicity:
1. Ex-vivo CAR-T: Ex-vivo activation often leads to T cell exhaustion, characterized by increased surface markers (e.g., PD-1, LAG-3), impairing persistence and function[13][16][17]. Common side effects include cytokine release syndrome (CRS) and immune effector cell-associated neurotoxicity syndrome (ICANS), resulting from excessive immune activation by ex-vivo engineered cells[8][8].
2. In-vivo CAR-T: In-vivo generated cells may reduce T cell exhaustion (e.g., via vector optimization to enhance mitochondrial respiratory function)[21][10] and lower systemic toxicity (e.g., CRS); the in-vivo environment naturally regulates immune responses, minimizing hyperactivation[8][8]. For example, animal models show in-vivo produced CAR-T cells achieve anti-tumor efficacy comparable to traditional ex-vivo CAR-T[10].
Technical Dependence and Limitations:
1. Ex-vivo CAR-T: Primarily effective for hematological cancers; limited efficacy in solid tumors due to microenvironmental suppression and physical barriers[17][22]. Its engineering methods are rigid, lacking flexibility[23][17].
2. In-vivo CAR-T: Flexible design (e.g., combining nanoparticles or exosomes) facilitates solid tumor targeting by overcoming tumor microenvironment barriers[12][4][24]. However, vector optimization is required to address uncontrollable in-vivo factors[9][3].
In summary, the core distinction of in-vivo CAR-T lies in "decentralized manufacturing" and "in-situ engineering," emphasizing convenience and biocompatibility[3][20].
III. Advantages of In-Vivo CAR-T
Based on research, in-vivo CAR-T offers significant advantages by addressing ex-vivo limitations:
Simplified Preparation and Reduced Costs: Eliminating expensive ex-vivo steps reduces production costs (e.g., lower equipment, personnel, and culture medium expenses); vectors (e.g., nanoparticles) enable mass production, improving scalability[18][20]. Studies confirm its ability to "bypass CAR-T manufacturing bottlenecks," making therapy more economical[18][8]. For example, lipid nanoparticle systems achieve efficacy comparable to traditional CAR-T in animal models at significantly lower costs[10].
Shorter Timeline and Improved Accessibility: Reducing preparation time from weeks to days enables timely treatment, particularly benefiting patients with progressive cancers[15][5][18][2]. Treatment can be administered in outpatient settings, reducing hospitalization needs and improving global accessibility[18][8].
Reduced Toxicity: Ex-vivo engineering often causes T cell exhaustion and hyperactivation, leading to CRS and other side effects. In contrast, in-vivo generated cells integrate more naturally into the immune system, lowering risks of systemic toxicity (e.g., CRS and neurotoxicity)[8][8][10]. For example, optimized vectors (e.g., sustained-release delivery) mitigate inflammatory responses in animal models[21].
Enhanced Functionality and Flexibility: Vector platforms support customizable designs (e.g., integrating immunomodulators) to boost CAR-T anti-tumor activity[4][13]. In-vivo T cells exhibit reduced phenotypic exhaustion (e.g., lower PD-1 expression), maintaining long-term tumor-killing capacity[13][12][21]. For solid tumors, targeted delivery (e.g., bioinstructive scaffolds) overcomes microenvironmental suppression[22][24].
Expanded Application Potential: As a "breakthrough technology," its low cost and simplicity facilitate development of new solid tumor therapies[25][17]. For example, combinations with exosomes or nanoparticles enable application across multiple cancer types[6][7][24].
In summary, the core advantages of in-vivo CAR-T—"simplified manufacturing, low cost, high accessibility, and reduced toxicity"—pave the way for widespread clinical application[20][3].
IV. Challenges of In-Vivo CAR-T
Despite promising prospects, in-vivo CAR-T faces significant technical hurdles:
Precise Targeting and Delivery Efficiency: The complex in-vivo environment requires vectors to specifically target T cells (avoiding other tissues) for efficient transduction. Current vectors (e.g., nanoparticles) suffer from off-target effects and low transduction rates; studies report uneven delivery in animal models[8][9][3]. For example, non-viral vector immunogenicity requires optimization to reduce side effects[12][9].
CAR Expression Stability and Persistence: Compared to ex-vivo engineering, in-vivo generated CAR-T cells may exhibit unstable or transient CAR expression; efficacy is affected by in-vivo immunosuppressive factors (e.g., M2 macrophages in the tumor microenvironment)[25][9][3]. Studies highlight the need for vector design (e.g., RNA-based constructs) to extend gene expression duration[20][10].
Safety and Toxicity Control: Vectors may induce toxicity (e.g., oncogenic risks with viral vectors) or unintended immune responses[8][9]. Optimizing in-vivo dosing to avoid hyperactivation remains challenging[8][3].
Technical Optimization and Lack of Standards: Absence of unified in-vivo engineering protocols results in variable efficacy across platforms (e.g., LNPs vs. viral vectors)[12][4]. Targeted delivery for solid tumors requires overcoming physical barriers and immunosuppression[17][22][24].
Scalability and Clinical Translation: Transitioning from animal models to human trials requires addressing scalability and standardized production[26][3]. Regulatory approval (e.g., FDA) for in-vivo therapies remains pending, indicating gaps in clinical validation[18][8].
These challenges reflect technical immaturity but are expected to be overcome with continued research[12][3].
V. Future Outlook
In-vivo CAR-T is regarded as the next generation of immunotherapy, with prospects rooted in technological evolution and application expansion:
Technological Innovation and Vector Development: Research focuses on optimizing delivery platforms, such as "multifunctional nanoparticles" or "engineered exosomes," to enhance targeting and safety[12][6][4]. For example, lipid-based vectors combined with immunomodulators (e.g., rapamycin) improve CAR-T mitochondrial function and persistence[21][10]. Novel "bioinstructive scaffolds" may enable local delivery to address solid tumor challenges[24][3].
Expanded Applications: Current in-vivo CAR-T focuses on hematological cancer models; future efforts will prioritize solid tumor treatment by overcoming microenvironmental barriers via targeted delivery[25][17][24]. Combination therapies (e.g., with selinexor or Axitinib) may enhance synergistic effects and reduce immunosuppression[13][27].
Accessibility and Industrialization: Simplified production will lower costs, transforming in-vivo CAR-T into a "versatile paradigm" for global patient access[12][20][15]. Biomaterial-based strategies may revolutionize CAR-T manufacturing[24].
Regulatory and Clinical Progress: Following animal model success (e.g., extended survival in leukemia models[10]), human clinical trials will advance, requiring adaptive regulatory frameworks[26][8].
Long-Term Vision: The goal is to transform CAR-T into an "off-the-shelf" therapy, revolutionizing cancer treatment via "bridging technological insights"[12][4]. Ultimately, in-vivo CAR-T may extend beyond oncology to treat autoimmune diseases[26][20].
In conclusion, in-vivo CAR-T represents a shift from "complex customization" to "convenient broad-spectrum therapy," poised to reshape immunotherapy[12][3].
Conclusion
In-vivo CAR-T therapy, centered on direct in-vivo engineering, addresses ex-vivo limitations via vector delivery systems, offering advantages such as low cost, high accessibility, and reduced toxicity. Despite technical challenges (e.g., delivery efficiency and safety), ongoing innovations (e.g., nanoparticle optimization and expanded applications) promise a bright future. In-vivo CAR-T is expected to become a mainstream cancer immunotherapy, advancing treatment democratization. While current progress relies on preclinical models, its "decentralized manufacturing" paradigm demonstrates transformative potential[18][12][20][3].
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