Engineered Lentiviral Vectors Enable Targeted In Vivo Generation of Functional CAR T and NK Cells

 

A novel engineered lentiviral vector platform has demonstrated the ability to safely and efficiently generate functional autologous chimeric antigen receptor (CAR) T and natural killer (NK) cells directly within the body following a simple intravenous injection, according to a new study published (Journal Pre-proof) in Molecular Therapy. This approach, developed by researchers at Interius BioTherapeutics, could transform the accessibility of CAR therapies for cancer and potentially autoimmune diseases by eliminating the need for complex, costly, and time-consuming ex vivo cell manufacturing and preconditioning chemotherapy.

Current Methods and Limitations in CAR Therapies

Autologous CAR T-cell therapies have revolutionized the treatment of B-cell malignancies, yet their broader adoption faces significant barriers. Manufacturing is complex: patient cells must be extracted, genetically engineered in specialized labs, expanded, and reinfused—a process that can take weeks, is costly, and varies in success. Patients also require toxic lymphodepleting chemotherapy to create “space” for the infused cells, which poses risks, especially for those with advanced disease or where chemotherapy is not standard care. These factors, combined with the personalized nature and complex logistics, limit patient access and drive up treatment costs.

Advantages of Lentiviral Vectors for Immune Cell Engineering

Lentiviruses, complex retroviruses from the Retroviridae family, can infect both dividing and non-dividing cells through active nuclear import enabled by viral accessory proteins. Their stable genomic integration and long-term gene expression make lentiviral vectors (LVs) leading tools for gene therapy. Modern third-generation LVs use split-packaging systems, remove pathogenic accessory genes (vif, vpr, vpu, nef), and employ self-inactivating (SIN) LTRs to lower oncogenic risk. Pseudotyping with vesicular stomatitis virus glycoprotein (VSV-G) broadens cell targeting. Together, these advances boost safety while maintaining high transduction efficiency in primary human cells.

LVs offer several advantages over alternative delivery systems. Stable genomic integration ensures durable CAR expression—a critical factor for sustained therapeutic effects. LVs consistently achieve high transduction efficiency, often exceeding 50% CAR expression in primary T and NK cells, generating enough functional cells for robust anti-tumor activity. Their ability to transduce non-dividing cells makes them ideal for modifying quiescent lymphocytes. With a large cargo capacity (8–10 kb), they accommodate complex CAR constructs and additional genetic elements. Importantly, they feature favorable safety profiles, including SIN designs and a lower risk of insertional mutagenesis than earlier gamma-retroviruses. Their clinical validation, exemplified by FDA-approved LV-based therapies like Kymriah®, further supports their use.

Rational Engineering of INT2104 for In Vivo CAR Generation

The new platform, embodied by the clinical candidate INT2104, directly addresses the limitations of conventional CAR T-cell therapies by enabling precise in vivo generation of CAR T and NK cells. INT2104 uses a rationally engineered, third-generation LV optimized for targeted delivery. Its core is the detargeted fusogen Gen 2.1, based on VSV-G but structurally modified with a key I182E mutation that abolishes LDL-R binding while preserving membrane fusion. Additional T214N and T352A mutations enhance serum stability, enabling safe IV delivery. For cell-specific targeting, the vector includes a high-affinity CD7 binder—a single-chain variable fragment (scFv) fused to an engineered Fc stalk that eliminates Fc receptor interactions—ensuring selective binding to CD7+ T and NK cells, including less-differentiated subsets. Once bound, INT2104 delivers a fully human CAR20 transgene, enabling the modified cells to target and destroy CD20+ B cells.

Mechanism: After IV administration, INT2104 particles bind CD7+ T and NK cells via the CD7 binder. The Gen 2.1 fusogen mediates fusion and nuclear delivery of the CAR20 transgene. The transduced cells then express the CAR20 protein on their surface, transforming them into functional CAR T or NK cells capable of recognizing and eliminating CD20+ malignancies.

Preclinical Evidence for Targeted In Vivo CAR T and NK Cell Generation

Specific Targeting In Vitro with Minimal Off-Target Transduction

Preclinical data strongly supports INT2104’s safety and efficacy. In vitro, INT2104 specifically transduced human CD4+ and CD8+ T cells and NK cells within PBMCs, generating CAR20+ cells at levels comparable to ex vivo manufacturing. These cells efficiently killed CD20+ B-cell tumor lines (Raji, Daudi) in an antigen-specific, dose-dependent manner, as confirmed by the lack of killing of CD20-knockout Raji cells. Upon target engagement, the engineered cells produced IFNγ, TNFα, and IL-2. Critically, INT2104 showed minimal off-target B-cell transduction—even at high vector doses—failing to transduce primary B cells from healthy donors or patients with mantle cell lymphoma, follicular lymphoma, or chronic lymphocytic leukemia, nor most B-cell lines. Notably, B cells engineered with a different vector to express CAR20 remained vulnerable to elimination by INT2104-generated CAR cells, further demonstrating safety.

Functional CAR Cells & B Cell Depletion In Vivo

In human CD34+ stem cell-engrafted NSG mice, a single IV dose of INT2104 generated CAR20+ CD4+ and CD8+ T cells detectable in blood, spleen, bone marrow, and liver within weeks, with near-complete depletion of human CD20+ B cells lasting over six weeks. In NSG mice expressing human IL-15 (which enhances NK cell engraftment), INT2104 also produced functional CAR20+ NK cells, resulting in similar B-cell depletion. In mice with established Raji tumors, a single IV dose post-engraftment led to complete tumor elimination within three to four weeks, with higher doses accelerating clearance.

Specific Targeting in Non-Human Primates

In cynomolgus macaques, IV administration of an INT2104 surrogate vector (modified for cross-species compatibility) showed targeted biodistribution to lymphoid tissues and a >50% drop in circulating CD20+ B cells within four days. Immunofluorescence confirmed CAR protein expression exclusively on target T cells (CD3+) in spleen and liver and NK cells (NKG2A+) in spleen, with no expression on B cells, macrophages, hepatocytes, or lung cells. Uptake by liver macrophages was observed but did not lead to CAR expression, likely due to innate restriction factors such as SAMHD1. These results underscore INT2104’s precision, efficacy, and safety for in vivo CAR generation.

Implications and Future Directions

INT2104 represents a major advance toward simplifying and expanding access to cell therapies. By enabling the body to produce its own CAR-equipped cells after a single IV infusion, it bypasses ex vivo manufacturing and preconditioning chemotherapy—offering the promise of a true “off-the-shelf” approach. Preclinical data strongly supports advancing INT2104 into clinical trials to confirm its safety, efficacy, and durability in patients.

Reference

Andorko JI, Russell RM, Schnepp BC, Grubaugh D, Mullen KF, Wakabayashi A, Carrington LJ, O’Malley T, Kuri-Cervantes L, Culp TD, Johnson PR, Targeted in vivo delivery of genetic medicines utilizing an engineered lentiviral vector platform results in CAR T and NK cellgeneration, Molecular Therapy (2025), doi: https://doi.org/10.1016/j.ymthe.2025.06.036

 

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1.  Development of a Pharmacokinetic (PK) Mouse Serum GLP ELISA for an Anti–CD19–AntiCD3 Diabody
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