Cell Therapy Goes Full-Drug: End-to-End Deconstruction of In Vivo CAR-T

I. What is CAR-T Cell Therapy?

CAR-T therapy refers to the ex vivo genetic engineering of patient-derived peripheral-blood T cells to express a chimeric antigen receptor (CAR) that specifically recognizes tumor-associated antigens. After activation and expansion in vitro, the CAR-T product is re-infused to eradicate malignant cells.

Since the first CAR-T approvals in 2017, the global market has expanded continuously: sales reached USD 2.014 billion in H1-2024 and are projected to hit USD 21.8 billion by 2030.

CAR architecture has undergone five generational iterations. All five generations share four conserved domains: extracellular antigen-binding domain, hinge, trans-membrane domain and intracellular signalling domain.

Evolution of CAR structure [1]

Functional comparison is summarized below:

Generation	Core structural hallmark	Functional advantage
1st	CD3ζ only; no co-stimulatory domain	Basic antigen recognition & T-cell activation; simplest structure
2nd	CD3ζ + 1 co-stimulatory domain (CD28 or 4-1BB)	Amplified activation signal; enhanced proliferation & persistence
3rd	CD3ζ + 2 distinct co-stimulatory domains (CD28-4-1BB or ICOS-4-1BB)	Further strengthened signalling; superior cytotoxicity & durability versus 2nd
4th	CD3ζ, co-stimulatory domain + transgenic cytokine (e.g. IL-12, IL-15)	Activation-triggered release of immunostimulatory cytokines; augmented tumour infiltration
5th	TCR-like activation, co-stimulatory & cytokine signalling in one universal CAR	Near-physiological cytokine signalling; comprehensive T-cell potentiation

II. End-to-End Roadmap of Ex vivo and In vivo CAR-T Pathways

1. Conventional Ex vivo CAR-T

• Patient screening & referral → Leukapheresis → T-cell activation (CD3/CD28, cytokines) → Gene transfer (lentiviral/γ-retroviral or non-viral) → Ex vivo expansion & QC release → Lymphodepletion (cyclophosphamide/fludarabine) → Infusion & monitoring

Pain Points & Bottlenecks

• Manufacturing complexity and long vein-to-vein time (2–7 weeks); bridging therapy often required for rapidly progressive disease
• Patient-specific products → batch-to-batch variability, unstable yields, high cost
• Lymphodepletion causes prolonged leukopenia and infection risk
• Entire workflow demands high GMP capacity, cold-chain logistics and multi-site coordination

2. In vivo CAR-T

• A single “off-the-shelf” formulation is injected directly into the patient; host T cells are genetically re-programmed in situ to express CAR and expand into functional cytotoxic populations

Major Implementation Routes

• i. Targeted viral vectors: engineered lentivirus/retrovirus or AAV whose envelope/capsid is pseudotyped and decorated with T-cell–directed ligands (scFv, nanobody, DARPin) to selectively transduce CD3/CD4/CD8 subsets
• ii. Non-viral nano-delivery: lipid nanoparticles (LNP) or biodegradable polymeric nanoparticles (e.g., PBAE) encapsulating mRNA or DNA; surface conjugation with anti-CD3/CD4/CD5/CD8 or lipid-composition tuning enhances T-cell uptake and expression

Key Attributes

• i. Scalable, standardized “one-product” inventory with immediate availability
• ii. Lymphodepletion can be omitted or minimized, preserving immune networks and favouring epitope spreading
• iii. mRNA formats give transient expression, allowing programmable repeat-dosing/dose-titration that balances efficacy and safety

Schematic of ex vivo and in vivo CAR-T workflows [1]

III. Technical Essentials for In vivo CAR-T

1. Vector/Delivery System & Targeting Strategy

Viral Vectors

• Lentivirus: pseudotyped envelopes (VSV-G, measles, NiV, Sindbis, cocal, etc.) plus T-cell-targeting ligands (anti-CD3/CD4/CD8 scFv/DARPin) enable selective tropism and membrane fusion; integrase activity supports durable CAR expression
• AAV: capsid surface-display of targeting ligands; episomal persistence provides medium-term expression without integration

Non-viral Nano-delivery

• LNP: ionizable cationic lipid + helper lipids (phospholipid/cholesterol/PEG-lipid); endosomal escape releases mRNA; antibody-conjugation further enhances T-cell uptake
• Polymeric NPs: deliver mRNA or DNA; peptide/antibody decoration improves specificity; can be combined with transposon systems for prolonged expression (safety liabilities require careful assessment)
• Exosomes: engineered exosomes (e.g., dendritic-cell-derived) surface-armed with anti-CD3 or anti-EGFR nanobodies

Delivery vectors

2. Nucleic-acid Construct & Expression Kinetics

• mRNA design: cap analogues, optimized UTRs (α-/β-globin 5′/3′UTRs), N1-methyl-pseudouridine, poly(A) tail length collectively enhance stability, translation and reduce innate sensing
• Co-delivery: mRNA can be packaged together with siRNA (e.g., PD-1 siRNA) to synchronously relieve immune suppression
• Expression window: mRNA-LNP gives transient expression (days), amenable to “repeat-dose → real-time dose/toxicity tuning → avoidance of exhaustion”; DNA/integrating platforms prolong expression but raise insertional mutagenesis and off-target transduction concerns

3. Specificity & Safety

Consequences of Off-target Transduction/Transfection

• Transduction of tumour cells → “epitope masking” (cis-binding renders antigen inaccessible)
• Transduction of suppressive immune subsets (e.g., Treg) may exacerbate disease
• Germ-line or stem-cell transduction (viral integration) poses oncogenic or heritable risks

Immunogenicity & Repeat Dosing

• Intrinsic immunostimulatory properties of LNP lipids and complement activation require management (pre-medication protocols validated for marketed LNP-siRNA can be adapted)
• Pre-existing neutralising antibodies to AAV or pseudotyped envelopes
• Balancing immune responses against mRNA-LNP during multi-dose regimens

4. Process & Dosing

• Route of administration: intravenous preferred; loco-regional or intracavitary injection considered for selected indications to increase local exposure
• Dose–exposure relationship: mRNA payload, LNP composition (lipid ratio/particle size/surface chemistry), UTR selection and ligand density collectively determine in-vivo T-cell transduction efficiency and expression durability
• Combination strategies: multi-target (parallel mRNAs or sequential vectors), inducible switches, or synthetic cytokine receptors

 

IV. Ex vivo vs In vivo CAR-T: Key Differences, Pros & Cons

Development/Manufacturing Model

• Ex vivo: patient-specific, complex, long lead-time, high cost, but product attributes are tightly controlled
• In vivo: single batch-scale formulation, greatly simplified manufacturing & release, rapid accessibility

Immunobiology

• Ex vivo: requires lymphodepletion to favour engraftment and expansion; ex-vivo culture may reduce stemness and drive exhaustion
• In vivo: preserves intact immune microenvironment, facilitates epitope spreading; transient mRNA + fractionated dosing mitigates continuous stimulation, lowers CRS/ICANS risk and enables “dose-to-effect” titration

Expression Persistence

• Ex vivo: integrating vector + expansion → long-term persistence
• In vivo: mRNA requires repeat dosing; integrating viruses can be long-term but demand higher specificity and genomic safety margins

Safety Focus

• Ex vivo: monitor insertional mutagenesis, clonal outgrowth, vector integration sites
• In vivo: additional concerns—off-target/germ-line transduction, tumour-cell epitope masking, systemic immunogenicity/anaphylaxis

Indications & Clinical Scenarios

• Ex vivo: established in B-cell malignancies
• In vivo: attractive for disorders requiring broad access and rapid availability (auto-immunity, infection, fibrosis); in oncology can serve as rapid bridging or complement to ex-vivo products

V. Application Landscape & Strategic Recommendations

• Haematological tumours: choose integrating virus or multi-dose mRNA based on relapse risk and depth of remission required
• Autoimmune diseases: CD19/BCMA B-cell depletion; mRNA-LNP preferable for “low-toxicity + adjustable repeat dosing” chronic management
• Infectious diseases (e.g., HIV, HBV): large-scale accessibility and avoidance of lymphodepletion are major advantages
• Fibrosis/tissue repair: transient FAP-CAR or similar for local immune-remodelling in heart failure or liver fibrosis

VI. Translational & Regulatory Considerations

• Viral vectors: demonstrate selective tropism, long-term follow-up of insertion sites/clonal expansion, mitigate germ-line risk
• Non-viral: control formulation consistency, dsRNA residuals, immunogenicity assessment, repeat-dose strategy
• CMC: scalable, batch-consistent processes; critical quality attributes (size/zeta-potential/encapsulation efficiency/ligand stability) must be quantified
• Companion diagnostics: track CAR-cell kinetics in blood/tissue, monitor B-cell/tumour burden, cytokine biomarkers (CRS/ICANS risk)

VII. Absin Products & Services

1. mRNA Synthesis Reagents

Cat. #	Product Name	Size
abs60690	High-Yield T7 Co-transcription RNA Kit (N1-Me-pUTP)	50 rxn
abs60152	Vaccinia Capping Enzyme	500/2 k/10 k U
abs60153	T7 RNA Polymerase	100/250 k U
abs60166	2´-O-Methyltransferase	2.5/10/50 k U
abs60539	DNase I	1/5 k U
abs60150	RNase Inhibitor	250 µL/1 mL

Reference: 1. Huang Y, Cao R, Wang S, Chen X, Ping Y, Zhang Y. In vivo CAR-T cell therapy: New breakthroughs for cell-based tumor immunotherapy. Hum Vaccin Immunother. 2025 Dec;21(1):2558403.