In Vivo CAR-T Efficacy Evaluation

In vivo CAR-T represents an emerging approach to adoptive cell therapy in which CAR constructs are delivered directly to target immune cells within the body — bypassing the ex vivo manufacturing steps required by conventional CAR-T. By generating cytotoxic CAR effector cells endogenously, this strategy reduces production complexity and broadens potential access compared to patient-derived cell therapies.

Mechanism

In vivo CAR-T delivery strategies can be broadly categorized into:

• Viral vectors (e.g. lentivirus, AAV)

• Non-viral systems (e.g. lipid nanoparticles (LNP))

For viral delivery systems, CAR transgenes can integrate into the host genome, enabling persistent expression and expansion together with proliferating T cells. Therefore, a single-dose administration design is typically adopted.

Aspect	Targeted Lipid Nanoparticle-mRNA (tLNP-mRNA)	Viral Vector
Duration	Transient (days to ~1-3 weeks; expression peaks at ~6-24 hours and declines rapidly thereafter)	Stable/long-term (weeks to years/lifelong due to genomic integration for lenti/retro, or episomal persistence for AAV)
Mechanism	mRNA translated directly in cytoplasm; no nuclear entry or genomic integration. Expression fades as mRNA degrades and CAR is diluted during cell division.	DNA payload integrates into host genome (lentivirus/retrovirus) or persists as episome (AAV) → continuous transcription from the promoter.
Level & Kinetics	High, transient expression; can achieve >45-69% CAR-positive T cells (splenic/circulating) after a single dose in vivo.	Consistent, stable expression level maintained over time; supports persistent CAR expression on progeny cells.
Clinical/Preclinical Implications	Ideal for "hit-and-run" control (e.g., titratable via re-dosing); reduces risks associated with chronic CAR signaling (e.g., T cell exhaustion, on-target/off-tumor toxicity).	Suited for durable responses (e.g., in hematologic cancers); offers "one-and-done" potential but is harder to control or turn off if severe toxicity occurs.

Our Advantages

20+ NCG immune reconstitution models
Well-established huPBMC and huHSC platforms with readily available inventory
One-stop solutions for both in vitro and in vivo evaluation

1,000+ oncology projects completed annually
Robust study design and extensive IND experience, delivering end-to-end support from strategy through execution to high-quality results

200+ high-quality PBMC donor batches
Enabling rapid positive efficacy screening and supporting customized study designs
300+ CDX models and 100+ luciferase-labeled cell lines

Rapid access to cell resources
Integrated target validation and tumorigenicity testing to accelerate study initiation
Flexible collaboration models tailored to client needs, including co-development opportunities

GemPharmatech routinely performs PBMC donor screening in both NCG and NCG-MHC-dKO mice. To date, the company has established a comprehensive resource bank comprising more than 200 PBMC donors and over 300 CDX models. For widely used tumor models, such as Nalm-6-Luc (CD19⁺), tumor engraftment and donor screening are continuously conducted to enable rapid responses to research and market needs.

Given the relatively long establishment timeline of huHSC-NCG models (>12 weeks), GemPharmatech maintains pre-established huHSC-NCG cohorts to significantly shorten study initiation timelines and accelerate the development of in vivo CAR-T therapeutics.

To accelerate the development of in vivo CAR-T therapeutics, robust and well-designed preclinical evaluation systems are essential. Leveraging advanced humanized mouse models and extensive non-clinical expertise, GemPharmatech is committed to supporting partners in overcoming key translational challenges and advancing innovative therapies toward the clinic.

Case Studies

Case Studies & Validation Data

Viral Delivery Systems

Single-Dose Efficacy of Integrating Viral CAR-T in NCG-MHC-dKO Mice
Figure 1. Single-dose efficacy of an integrating viral CAR delivery system in NCG-MHC-dKO mice.

(A) NCG-MHC-dKO mice were engrafted with human PBMCs (D-1) and inoculated with Nalm6-Luc tumor cells (D-4). A single i.p. dose of test article (1E8 TU) or vehicle was given on D0. (B) The test article group showed sustained tumor regression as measured by bioluminescence imaging (BLI) and (C) prolonged survival compared with the vehicle control. (D) BLI images of live animals.
As shown in Figure 2C, NCG-MHC-dKO mice, where MHC class I and II has been knocked out, demonstrate a lower incidence of graft-versus-host disease (GvHD) following hPBMC transplantation, and the therapeutic window is extended markedly (≥80 days). This makes NCG-MHC-dKO an ideal model for evaluating in vivo CAR-T therapeutics.

LNP Delivery Systems

Multi-Dose Efficacy of Transient LNP-CAR in NCG-MHC-dKO Mice
Figure 2. Multi-dose efficacy of LNP-based transient CAR delivery system in NCG-MHC-dKO mice

(A) NCG-MHC-dKO mice were engrafted with human PBMCs (D-14) and inoculated with Nalm6-Luc tumor cells (D-8). Five doses (Q3D, 30 μg each) of vehicle or LNP-CAR mRNA test articles were administered intravenously. (B) Tumor growth was suppressed (Test article 1 vs Vehicle on D18: p<0.05; Test article 2 vs Vehicle on D18: p<0.001). (C) Animal survival was prolonged compared with the vehicle group. (D) Representative bioluminescence images of live animals.
In LNP-based delivery systems, CAR-encoding mRNA is delivered into the cytoplasm and subsequently translated into CAR protein through transient mRNA expression. This approach relies on the presence and basal abundance of target immune cells, typically T cells. Therefore, multiple administrations are generally required, and adequate immune cell reconstitution at the time of dosing is critical. In practice, dosing is usually initiated only after human CD3+ T cells reach a predefined threshold level.

In Vivo Toxicity and Safety Evaluation

In Vivo CAR-T: huHSC-NCG Model
The huHSC-NCG mouse model is generated by transplanting human CD34⁺ hematopoietic stem cells (HSC) into sublethally irradiated immunodeficient NCG mice. This model enables the endogenous reconstitution of functional human immune cells, including T cells and immature B cells, with stable engraftment and extended survival period (>90% survival beyond 35 weeks). Owing to its robust and durable human immune system reconstitution, the huHSC-NCG model is widely applied in studies of B cell depletion, in vivo CAR-T biodistribution, as well as long-term toxicity and safety evaluation of in vivo CAR-T therapeutics.

Figure 3. Immune cell profiling in huHSC-NCG mice

Figure 4. NCG mouse survival after huHSC reconstitution

This model was applied successfully in the toxicity and safety evaluation of SYS6055, an in vivo CAR-T therapeutic developed by CSPC Pharmaceutical Group. In addition, the Center for Drug Evaluation (CDE) of the National Medical Products Administration has recognized PBMC- and HSC-humanized NCG mouse models as appropriate platforms for the non-clinical evaluation of in vivo CAR-T therapeutics, including studies conducted with clinical-grade materials.