Revolutionizing cancer treatment: the emerging potential and potential challenges of in vivo self-processed CAR cell therapy

Abstract

Chimeric antigen receptor (CAR) cell immunotherapies, including CAR-T, CAR-Macrophages, CAR-Natural Killer, CAR-γδ T, etc., have demonstrated significant advancements in the treatment of both hematologic malignancies and solid tumors. Despite the notable successes of traditional CAR cell manufacturing, its application remains constrained by the complicated production process and expensive costs. Consequently, efforts are focused on streamlining CAR cell production to enhance efficacy and accessibility. Among numerous proposed strategies, direct in vivo generation of CAR cells represents the most substantial technical challenge, yet holding great promise for achieving clinical efficacy. Herein, we outlined the current state-of-the-art in vivo CAR therapy, including CAR technology development, transfection vectors, and influence factors of construction of CAR in vivo. We also reviewed the types and characteristics of different delivery systems and summarized the advantages of in vivo CAR cell therapy, such as rapid preparation and cost-effectiveness. Finally, we discussed the limitations, including technical issues, challenges in target and signal design, and cell-related constraints. Meanwhile, strategies have correspondingly been proposed to advance the development of CAR cell therapy, in order to open the new horizons on cancer treatment.

Keywords: CAR; Construction; Delivery technology; Immunotherapy; Vector particles.

Figure 1

Ex vivo vs. in vivo CAR cell therapies: a comparative. (A) The ex vivo approach begins with the isolation of immune cells from the patient's blood. These cells are then activated, expanded, and genetically modified in a controlled laboratory environment. Following stringent quality control measures, the engineered CAR cells are reinfused into the patient. (B) In contrast, the in vivo method involves directly infusing vector particles (represented as red dots) into the patient. These vectors interact with the patient's immune cells within the body, selectively transferring the genetic material necessary to encode the CAR.

Figure 2

The development process of CAR cells from the first to the fifth generation: CAR cells are categorized into five generations, each defined by distinct intracellular signal transduction structures.

Figure 3

The mechanisms of CAR-T, CAR-NK, CAR-M, and CAR-γδ T cell therapies. (A) When CAR-T cells bind to target antigens, they activate intracellular signaling pathways like PI3K/AKT/mTOR, MAPK/ERK, and NF-κB, leading to T cell proliferation, cytokine production, and cytotoxic activity through perforin and granzyme release, inducing cancer cell apoptosis. CAR-T cells also secrete cytokines such as IFN-γ and IL-2, boosting the antitumor response, with some differentiating into memory T cells for long-term immune surveillance and reduced cancer recurrence. (B) Similarly, activated CAR-NK cells produce perforin and granzyme, promoting cancer cell death through caspase-mediated apoptosis. CAR-NK cells express receptors such as KIRs, NKG2D, and DNAM-1, and mediate antibody-dependent cellular cytotoxicity (ADCC) via CD16, playing a crucial role in targeting HER2 and EGFR in solid tumors. Their efficient ADCC is linked to better outcomes in various cancers. (C) CAR-M cell therapy uses engineered macrophages to enhance phagocytosis and antigen presentation, adapting to tumor environments while promoting pro-inflammatory signaling and suppressing tumor-promoting polarization. CAR-M cells secrete IFN-γ, recruiting and activating immune cells, and upregulate MHC-I and MHC-II, improving T cell activation and immune infiltration into tumors. (D) CAR-γδ T cells exhibit antitumor effects via TCR, NKRs, and CD16, triggering ADCC and directly killing tumor cells by releasing TRAIL, FasL, perforin, and granzyme. They enhance cytotoxic T cell and NK cell functions through IFN-γ, TNF, and CD137 signaling, and produce GM-CSF to regulate dendritic cell infiltration, further augmenting the antitumor immune response.

Figure 4

Delivery strategies for in vivo CAR construction. The primary vectors for viral transduction (left) include lentiviral vectors, adeno-associated viral vectors, and retroviral vectors. Non-viral transduction (right) vectors consist of lipid nanoparticle vectors, gel vectors, and other nanoparticle vectors like plasmid vectors (Sleeping Beauty, minicircles, etc.).

Figure 5

The Vector entry modes into the cell. (A) LVs contain one or more viral glycoproteins and two copies of a single-stranded RNA (ssRNA) genome encapsulated within a nucleocapsid. Once inside, the transferred gene undergoes reverse transcription, is transported into the nucleus, and integrates into the host genome. (B) After internalization, AAVs are enclosed in endocytosed vesicles. The acidification of these vesicles triggers viral escape into the cytoplasm, where the viruses utilize the cellular microtubule transport system to approach the nucleus. AAVs then interact with the nuclear pore complex to gain entry into the nucleus. Once inside, the single-stranded DNA undergoes conformational changes to form double-stranded DNA, which may subsequently integrate into the host genome. (C) Retroviruses bind to host cell surface receptors via their glycoproteins. Subsequent membrane fusion introduces the viral capsids into the cells. In the nucleus, the capsids uncoat, and the viral RNA is reverse transcribed and integrated into the host genome. (D) In synthetic vectors, CAR-encoding nucleic acids are complexed with NPs, LNPs, or gel-based carriers. After escaping the endosome, mRNA payloads are available for translation, while packaged DNA may reach the nucleus for potential integration into host chromatin.

Figure 6

Crucial factors to consider for the application of CAR cells. 1) Targeting and biodistribution are crucial for optimizing therapeutic efficacy and advancing novel technologies. 2) CAR immunogenicity is a significant concern due to its diverse origins and multifaceted impact on treatment outcomes. 3) Safety and control-related challenges are of particular concern in in vivo CAR technology, including cytokine release syndrome (CRS), neurotoxicity, and potential insertional mutations during gene delivery. These may impair normal cellular function and induce tumorigenesis.

Figure 7

The comprehensive conclusion for CAR cell construction in vivo. This comprehensive summary covers the critical factors in constructing CAR cells, including gene editing, transfection techniques, vector selection, cell types, and CAR structure. It also illustrates the related elements of CAR cell construction and their interrelationships.