In Vivo CAR T Cell Therapies

Chimeric antigen receptor (CAR) T-cell therapy transformed the treatment of several hematologic malignancies by redirecting a patient’s T cells to recognize and kill target cells. All currently approved CAR T products are made ex vivo: T cells are collected by apheresis, engineered and expanded in a manufacturing facility, and then reinfused. This paradigm is effective but costly, logistically complex, and time-consuming.

In vivo CAR T aims to solve these bottlenecks by engineering T cells inside the patient’s body. Instead of shipping cells to a plant, a one-time infusion of a vector or nanoparticle delivers genetic instructions that program circulating T cells to express a CAR. If successful, in vivo approaches could reduce time-to-treatment from weeks to days, broaden access, and enable indications beyond cancer, including autoimmunity and fibrosis. Recent reviews and perspectives highlight this shift from cell “product” to in-body genetic programming as a major frontier for immuno-gene therapy.

While ex vivo and in vivo CAR T share the same end goal, CAR expression on T cells, the biological and engineering steps differ:

1. Targeted delivery to T cells. In vivo platforms must deliver nucleic acids (DNA or mRNA) or vectors to T-cell subsets with high specificity and efficiency while minimizing off-target transfection (e.g., liver). Strategies under investigation include:
   • Targeted lipid nanoparticles (tLNPs) decorated with anti-CD5 or anti-CD8 binders to preferentially transfect T cells with an mRNA payload encoding a CAR. Mouse studies demonstrate selective CD8⁺ T-cell transfection and favorable tolerability compared with generic LNPs.
   • Viral vectors (e.g., LV/γ-retroviral or AAV derivatives) administered intravenously with tropism for lymphocytes, delivering the CAR cassette directly in vivo.
2. Genetic payload and expression kinetics.
   • mRNA payloads (LNP-based) drive transient CAR expression (days to weeks), which could be advantageous when reversible activity is desired (e.g., in autoimmune indications) or to mitigate prolonged toxicity. Preclinical data show in vivo–generated CAR T cells can expand and mediate cytotoxicity against antigen-positive targets.
   • DNA or integrating vectors aim for durable CAR expression, more akin to ex vivo products, but require careful control of insertional risk and expression levels.
3. Pharmacology and immunology. After successful delivery, in vivo–engineered T cells activate, expand, and kill target cells via CAR-mediated recognition (e.g., CD19 or CD20 on B cells). Pharmacodynamic readouts include B-cell aplasia (for B-cell targets), cytokine profiles, and T-cell kinetics. Early human protocols are also exploring preconditioning-free regimens, banking on in vivo transfection efficiency to obviate lymphodepletion.

The data in this graph was taken from the Beacon Cell Therapy Database

• The dominant viral delivery approach is Lentiviral, while Lipid Nanoparticles lead the way for non viral delivery methods.
• The growing number of non-viral, nanoparticle based-approaches highlights an ongoing shift towards safer, more flexible alternatives to traditional viral vectors.

A rich preclinical literature underpins in vivo CAR T development:

• tLNP–mRNA systems have shown preferential T-cell transfection and generation of functional CAR T cells in mice, with evidence of tumor clearance in xenograft models and reduced off-target exposure compared with benchmark LNPs. These studies emphasize the importance of ionizable lipid chemistry (e.g., LNP “L829”), antibody targeting (anti-CD5/anti-CD8), and payload design.
• Blood (ASH) preclinical presentations have described in vivo reprogramming of circulating human T cells using LNP-mRNA encoding second-generation CARs (e.g., anti-CD22), demonstrating transfection efficiency and functional activity in vitro and in vivo.
• Mechanistic and translational reviews in 2024–2025 outline opportunities and risks: controllable expression (mRNA), vector immunogenicity, anti-drug antibodies to targeting moieties, insertional concerns for integrating vectors, and the need for precise dose–exposure–response modeling specific to gene delivery pharmacology.

Although ex vivo CAR T trials number in the thousands, in vivo CAR T is only now entering the clinic. Two visible programs illustrate the clinical landscape as of October, 2025:

• Capstan Therapeutics – CPTX2309 (anti-CD19, tLNP-mRNA; Autoimmunity).
  • Design: First-in-human, Phase 1, healthy volunteer study assessing safety, tolerability, PK/PD of an intravenous in vivo CAR T candidate that transiently programs CD8⁺ T cells to express anti-CD19 CAR.
  • Rationale: Enable reversible B-cell depletion without apheresis, viral vectors, or chemotherapy; envisioned for B-cell–mediated autoimmune diseases. Company communications report first participants dosed and emphasize the potential for preconditioning-free administration.
• Interius BioTherapeutics – INT2104 / INVISE (CD20-targeted; Oncology).
  • Design: Phase 1 (INVISE) testing a first-in-class in vivo CAR gene therapy designed to generate CAR-T and CAR-NK cells targeting CD20 for relapsed/refractory B-cell malignancies. The trial, first opened outside the EU, received Paul Ehrlich Institute (PEI) approval in January 2025 to expand into Europe—the first such in vivo CAR gene therapy trial cleared in the region.

Landscape reviews from 2024–2025 note that multiple first-in-human studies are starting across platforms (tLNP-mRNA and viral), with interest from major pharmas and strategic deals, and suggest in vivo CAR T will be a key theme through 2025.

Opportunities:

• Speed and access. Avoiding apheresis/manufacturing queues could compress vein-to-treat timelines and expand global access.
• Cost and scalability. Centralized drug-like manufacturing (e.g., LNP batches) may lower per-patient costs and simplify supply chains.
• New indications. Transient CAR expression via mRNA could suit autoimmune disease, where reversible B-cell depletion is desirable; similarly, local or short-lived activity could be explored in fibrosis or organ-specific conditions.

Challenges:

• Targeting specificity and efficiency. Achieving high, selective delivery to T cells in humans at tolerable doses remains a central technical hurdle.
• Safety management. Traditional CAR toxicities (CRS, ICANS) still apply; systemic delivery introduces LNP/vector-specific risks (infusion reactions, complement activation, pre-existing anti-capsid antibodies). Careful dose escalation and stopping rules are key.
• Immunogenicity to the delivery vehicle. Re-dosing may be limited by anti-LNP antibodies or anti-vector immunity; strategies include shielded chemistries, alternative lipids, or transient immunomodulation.
• Durability vs control. mRNA systems offer control and reversibility but may require repeat dosing; integrating vectors improve persistence but raise genotoxicity concerns that must be mitigated by vector design and dosing.

1. Platform optimization:
   • Next-gen tLNPs with improved endosomal escape, refined ionizable lipids, and human T-cell–specific targeting ligands are likely to raise efficiency while reducing dose.
   • Programmable payloads (e.g., switchable CARs, logic-gated constructs, or mRNA cocktails enabling co-delivery of cytokines or dominant-negative receptors) to widen the therapeutic window.
2. Indication expansion:
   • Autoimmunity: Transient anti-CD19/CD20 in vivo CAR T could offer steroid-sparing and B-cell reset strategies across SLE, rheumatoid arthritis, and related conditions; early clinical steps (healthy volunteers) are under way.
   • Oncology: In vivo CD20 and CD19 programs will test whether disease-modifying efficacy comparable to ex vivo products can be achieved with more “drug-like” administration.
3. Regulatory and clinical science:
   • Expect fit-for-purpose guidance around manufacturing characterization of delivery vehicles (analogous to gene therapy CMC) and transfection/PD biomarkers as surrogate endpoints in early phases.
   • Comparative effectiveness vs existing ex vivo therapies will require well-designed patient trials, not only healthy volunteer safety studies.
4. Ecosystem and deal-making:
   • Analysts and trade coverage anticipate rapid pipeline growth through 2025, with cross-industry alliances (e.g., big pharma with in vivo platform biotechs) and selective M&A accelerating development.