Overview

On October 10, 2025, BMS acquired in vivo CAR-T company Orbital Therapeutics for $1.5 billion;

On August 21, 2025, Gilead acquired in vivo CAR-T company Interius BioTherapeutics for $350 million;

On June 30, 2025, AbbVie acquired in vivo CAR-T company Capstan Therapeutics for $2.1 billion;

On March 17, 2025, AstraZeneca acquired EsoBiotec for $1 billion, entering the in vivo CAR-T field.

With continuous investments from major multinational corporations (MNCs), the in vivo CAR-T field has demonstrated its platform potential and market value.

In vivo CAR-T therapy leverages mRNA-LNP delivery systems to generate CAR-T cells directly within the patient. Hzymes provides GMP-grade T7 RNA polymerase, modified nucleotides, and LNP formulation reagents to support in vivo CAR-T development.

Ex vivo CAR-T vs. In vivo CAR-T

CAR-T refers to a type of immunotherapy in which a patient’s own T cells are genetically engineered to express chimeric antigen receptors (CARs) that specifically recognize tumor-associated antigens on cancer cells, activating T-cell–mediated cytotoxicity.

The ex vivo CAR-T workflow involves:

cell collection and separation (obtaining leukocytes from peripheral blood and isolating T cells),

T-cell activation (using anti-CD3/CD28 antibodies),

viral transduction (delivering CAR genes via viral vectors),

in vitro expansion (culturing to amplify T cells),

and reinfusion (returning quality-controlled CAR-T cells to the patient).

However, this process is lengthy, costly, highly individualized, and shows limited efficacy against solid tumors.

The emerging in vivo CAR-T approach, in contrast, uses delivery vectors (such as LNPs or lentiviruses) to directly introduce CAR genes into endogenous T cells in vivo, effectively reprogramming them into CAR-T cells within the body. This eliminates steps like T-cell isolation, in vitro expansion, and lymphodepletion, reducing inter-patient variability and showing promise for solid tumor applications.

Ex vivo CAR-T vs In vivo CAR-T

Pathways to Achieve In vivo CAR-T: mRNA-LNP and Lentiviral Vectors

The key challenge of in vivo CAR-T lies in targeted delivery—how to precisely deliver CAR genes to T cells and achieve specific expression that activates their tumor-killing ability.

Currently, two main approaches are being explored: antibody-conjugated lipid nanoparticles (Ab-LNPs) and lentiviral vectors. These two platforms complement each other in terms of delivery mechanisms and expression characteristics, jointly advancing the development of in vivo CAR-T technology.

In vivo CAR-T Delivery Approaches

The mRNA-LNP Approach: A “Transient Expression System” for In Situ T-Cell Reprogramming

The mRNA-LNP approach uses antibody-conjugated LNPs (Ab-LNPs) to deliver CAR mRNA precisely into T cells in vivo, enabling in situ reprogramming.

Its core lies in Ab-LNP targeting modifications and multifunctional CAR molecular design, where antibodies bind to T-cell–specific surface receptors, overcoming the challenge of nonspecific delivery.

Preparation of Ab-LNPs and In Vivo CAR-T Expression

Preparation of Ab-LNP and In Vivo CAR-T Expression

01. Ab-LNP Targeting Mechanism: The “Molecular Bridge” for T-Cell Specificity

Through antibody modification, a specific “LNP–T-cell receptor” targeting mechanism is constructed:

Targeted enrichment: 

The surface of Ab-LNPs is conjugated with T-cell–specific antibodies (such as anti-CD3 or anti-CD8), which bind to receptors on circulating or splenic T cells and help avoid clearance by the liver’s reticuloendothelial system (RES).

Receptor-mediated endocytosis: 

The antibody–receptor interaction triggers endocytosis, internalizing Ab-LNPs into the T cell.

mRNA release: 

The ionizable lipid becomes protonated in the acidic endosomal environment, generating a proton sponge effect that disrupts the endosome and releases mRNA into the cytoplasm for efficient CAR protein expression.

02. Ab-LNP Structural Design: Synergistic Optimization of Four Components

Ab-LNPs rely on the coordinated design of antibodies, lipids, and helper components, all finely tuned for T-cell targeting and mRNA protection.

Surface Modification of LNP Determines Targeting Function

LNP surface modifications determine targeting functionality:

Targeting antibody: 

Should exhibit high specificity, low immunogenicity, and optimal affinity, typically conjugated via a PEG spacer arm. Common antibodies include anti-CD3 (pan-T-cell targeting), anti-CD8 (cytotoxic T-cell targeting), anti-CD28 (dual targeting and co-stimulation), and anti-Treg for regulatory T cells.

Ionizable lipid: 

pKa 6.2–6.5 (neutral in blood, protonated in endosomes); hydrophobic tails with mixed C14–C18 chains enhance T-cell membrane fusion; head groups with substituted amines (e.g., tetrahydroisoquinoline) improve π–π stacking with mRNA for stable complex formation.

Helper lipid and cholesterol: 

Helper lipids adjust membrane fluidity, while cholesterol stabilizes the nanoparticle structure. When the molar ratio of ionizable lipid:cholesterol ≥ 1:1, Ab-LNPs retain >90% encapsulation efficiency after 48 hours in plasma at 37 °C; reducing the ratio to 1:0.8 causes a sharp drop to 65%.

PEG-lipid: PEG2000-DMG 

forms a hydrated surface layer to extend circulation time and prevent the accelerated blood clearance (ABC) effect. Clinically, PEG-lipid content is typically 1–2% (molar) with biodegradable ester-linked PEG chains.

03. Key Process Parameters: The “Precision Scale” Determining Transduction Efficiency

The preparation of Ab-LNPs requires tight control of process parameters, as deviations can cause loss of targeting or increased toxicity.

04. Preclinical and Clinical Validation: From Animal Models to Human T-Cell Activation

Hematologic malignancies: 

In a joint study by the University of Pennsylvania and Capstan, CD8 antibody–modified Ab-LNPs delivering CD19 CAR mRNA achieved remarkable outcomes in leukemia mouse models:

1. Splenic CD8⁺ T-cell CAR expression rate: 68% (vs. 5.2% with unmodified LNPs);
2. 90% clearance of peripheral B cells (tumor-mimicking targets) within 24 hours;
3. In the high-dose group (2 mg/kg), complete tumor regression was observed in all three mice, with no liver toxicity.

Autoimmune diseases: 

In clinical trial HN2301, Prof. Zhu Chen’s team used CD8 antibody–modified Ab-LNPs with 22 antibodies per particle, ionizable lipid:mRNA ratio 10:1, and 96% encapsulation efficiency.

1. Among 5 patients with refractory lupus, high-dose infusion induced CAR expression in peripheral CD8⁺ T cells within 6 hours, sustaining B-cell clearance for 10 days.
2. After 3 months, the SLEDAI-2K score dropped from an average of 18 to 6 in 4 patients, with only mild (grade 1) CRS and no neurotoxicity.

Solid tumor studies: 

Academician Weihong Tan’s group developed dual-function Ab-LNPs co-delivering CAR mRNA and CCL21 mRNA.

1. In a melanoma mouse model, CCL21 guided CAR-T infiltration, increasing intratumoral CAR-T counts 3.8-fold compared with standard Ab-LNPs.
2. When combined with PD-1 inhibitors, tumor suppression reached 89% with no systemic toxicity, addressing the major challenge of poor CAR-T infiltration in solid tumors.

Product Information

Bibiliography

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