Overview of In Vivo CAR-T Therapy
The Development Background of In Vivo CAR-T Cell Therapy
Conventional ex vivo CAR-T manufacturing involves multiple steps: leukapheresis, T-cell sorting
and activation,
lentiviral or retroviral
transduction, CAR-T cell expansion, release testing, product transportation, and final infusion.
This process typically takes 1–3 weeks.
Due to its high cost, lengthy and complex procedures, and potential production failures,
conventional autologous CAR-T therapy has limited
patient accessibility. These limitations have motivated the exploration of alternative
strategies.
Allogeneic CAR-T (Universal CAR-T) therapies were developed to address these constraints but
introduce immune rejection risks,
such as
graft-versus-host disease (GVHD). In contrast, in vivo
CAR‑T therapy delivers CAR-encoding transgenes directly into
endogenous T cells, reprogramming them in situ. This approach greatly simplifies the process,
reduces costs, and represents a
promising strategy to overcome the limitations of conventional CAR-T therapies.
Figure 1. Comparison of In Vivo and Ex Vivo CAR-T Therapy Processes[1].
Table 1. Comparison of the characteristics of different types of CAR-T cells[2].
| Dimension |
Traditional CAR-T |
Universal CAR-T |
In vivo CAR-T |
| Cell source |
Isolation of autologous T cells and in vitro expansion |
PBMCs from healthy donors; iPSC |
Editing in vivo in patients |
| Preparation time |
3–6 weeks to complete product preparation and re-infusion |
Preparation in advance and immediate infusion |
Preparation in advance, immediate administration, 10–17 days to reach the peak
amplification |
| Relative cost |
High (may decrease in the future) |
Moderate |
Low |
| Clinical toxicity manifestations |
CRS; ICANS; hematotoxicity; potential long-term side effects (Secondary infection;
SPMs) |
GVHD; CRS; ICANS; hematotoxicity; Secondary infection |
CRS; ICANS; hematotoxicity; Secondary infection |
| Persistence |
Intermediate to long (months to years) |
Short to intermediate (weeks to months) |
Unknown (insufficient data) |
| Phenotypic control ability |
High, specific phenotypes can be induced by in vitro preconditioning |
High, specific phenotypes can be induced by in vitro preconditioning |
Low, limited ability to control phenotype in vivo |
| Technology maturity |
High, multiple products have already received market approval |
Low, still in clinical studies |
Low, still in clinical studies |
CAR: chimeric antigen receptor, CRS: cytokine release syndrome, GVHD: graft versus host disease,
ICANS: immune effector cell-associated
neurotoxicity syndrome, iPSC: induced pluripotent stem cell, SPMs: secondary primary
malignancies.
The Advantages and Characteristics of In Vivo CAR-T Therapy
Since 2017, in vivo CAR-T therapy has progressed from proof-of-concept studies using synthetic
DNA nanocarriers to optimized viral and non-viral
vectors, with preclinical validation in hematological and solid tumor models, and has entered
early clinical trials for hematological
malignancies and autoimmune diseases. Beyond this rapid development, in vivo CAR-T therapy has
demonstrated several practical advantages
over conventional adoptive approaches, including streamlined treatment workflows, accelerated
therapeutic delivery, and improved patient
accessibility (Figure 2).
Figure 2. In vivo CAR-T vs. adoptive therapy: streamlining process and improving
accessibility[2].
1. Streamlining manufacturing and therapeutic processes
In adoptive therapy, CAR-T treatment generally takes about 10–25 days. In vivo CAR-T therapy
directly generates immune cells using CAR
carrier drugs without complex in vitro expansion or individualized operations, significantly
simplifying the treatment process.
It eliminates the need for lymphodepletion chemotherapy and repeated quality checks. This
shortens the time from treatment decision
to drug administration, improving overall treatment efficiency.
2. Reducing costs and improving patient access
By eliminating individualized production and long-cycle in vitro operations, in vivo CAR-T
enables "off-the-shelf" products, significantly reducing
costs and improving patient accessibility. At the same time, it allows for the potential of
large-scale production and broader clinical application.
3. Efficacy and safety advantages
The main procedural difference between adoptive and in vivo CAR-T therapy lies in
lymphodepletion pretreatment. While pretreatment in adoptive
therapy helps CAR-T cell adaptation and maximizes therapeutic benefits, it carries risks such as
infection, leukopenia, and pulmonary
vein occlusive disease. In contrast, in vivo CAR-T relies on endogenous T cell generation,
preserving the complete immune system,
which supports a broad antitumor response and reduces the likelihood of antigen escape. Although
in vivo CAR-T may face efficacy limitations,
such as immune-mediated CAR-T clearance, it may provide advantages in stemness and
proliferation, resulting in superior in vivo
activity at lower doses.
Platforms and Strategies for In Vivo CAR-T Cell Production
The development of in vivo CAR-T therapy depends on advances in gene delivery and editing.
Unlike adoptive therapy, the therapeutic outcome
in this approach is determined during the in vivo generation process, which relies on the
precision, efficiency, and accuracy of CAR gene
delivery. Consequently, the selection of an appropriate delivery vector and strategy becomes
a critical consideration in the manufacturing
process. To date, three main technical platforms have been employed for in vivo CAR-T
generation, each offering distinct characteristics
and advantages.
Table 2. Comparison of three platforms of CAR-T cell generation[2].
| Dimension |
Virus-based vector platforms |
Nanoparticle-based carrier platforms |
Implant platform |
| Platform carrier type |
Lentivirus; Retrovirus; Adeno-associated virus
|
LNPs with different formulations |
Implants containing CAR virus vectors |
| Immunogenicity |
High, limit repeat dosing |
Low, supporting repeat dosing |
Low, use base material with high biocompatibility |
| Potential risk |
Off-target delivery; Non-targeted toxicity; Genomic integration error |
Off-target delivery; Non-targeted toxicity; Dose-dependent toxicity |
Non-targeted toxicity; Genomic integration error |
| Delivery efficiency |
Targeted clear, but limited packaging capacity |
Antibody modification enables precise targeting and packaging of large mRNA fragments |
In-scaffolds delivery |
| In-vivo editing efficiency |
Continuous expression, but may increase the risk of off-target |
Transient expression, persistence may be limited |
In-scaffolds editing with high efficiency |
| Technical progress |
Preliminary clinical trial results were obtained |
Clinical safety testing based on healthy volunteers |
Pre-clinical stage |
CAR: chimeric antigen receptor, LNPs: lipid nanoparticles.
In Vivo CAR-T Generation Based on Viral Vectors
Lentiviral vectors and adeno-associated viruses (AAV) are the most commonly used viral
vectors. Lentiviral vectors, capable of stable gene
integration and widely used in ex vivo CAR-T, are promising for in vivo delivery. However,
their in vivo application still faces several
challenges, such as host immune responses that may clear the virus, difficulties in
specifically targeting T cells, and the potential tumor
risk associated with random genomic integration.
To enhance the efficiency and safety of in vivo CAR-T generation, researchers have developed
various optimization strategies. For example,
some lentiviral platforms display multi-domain fusion proteins (MDFs) on the viral surface,
simultaneously providing T cell activation
signals and co-stimulatory signals, thereby improving T cell transduction efficiency. Other
studies focus on targeting specific T cell
subsets (such as CD4+ or CD8+ T cells), with the resulting CAR-T cells demonstrating
enhanced persistence and antitumor activity in vivo.
AAVs have also been explored, successfully reprogramming T cells and demonstrating antitumor
effects in models.
Despite promising preclinical results, genomic integration risks must be carefully monitored
to avoid potential malignant transformation.
Thus, viral vectors hold great potential, but safety and controllability remain key
considerations.
In Vivo CAR-T Generation Based on Nanoparticles
Nanoparticle (NP) platforms, mainly lipid nanoparticles (LNPs), offer non-viral gene
delivery for in vivo CAR-T cell generation. Compared to
viral vectors, nanoparticles are more readily engineered to target T cells, offer improved
safety profiles, and are relatively
cost-effective. Moreover, mRNA delivery avoids risks associated with genomic integration.
They are also suitable for large-scale
production, transport, and storage. LNPs typically contain ionizable lipids, phospholipids,
cholesterol, and PEGylated lipids,
with surface modifications enhancing T-cell targeting and delivery efficiency.
Early studies explored biodegradable polymeric nanocarriers and transposon systems for in
vivo CAR-T cell engineering. Although initial efficiency
was limited, these studies demonstrated the feasibility of in situ CAR-T generation. The
mRNA-LNP technology advanced by COVID-19 vaccines
has opened new opportunities for immune cell therapy. For example, CD5-targeted LNPs
delivering CAR mRNA successfully edited T cells and
cleared fibroblasts in mouse models, improving cardiac function. Strategies that co-express
CARs and
cytokines have further enhanced
therapeutic efficacy. The latest targeted LNP (tLNP) technologies further reduce off-target
delivery to the liver and have shown significant
antitumor or B-cell depletion effects in humanized leukemia mouse models and non-human
primates, along with favorable safety profiles.
To address limitations such as mRNA persistence and delivery efficiency, researchers have
also investigated circular RNA (circRNA) and DNA
platforms. CircRNA, due to its circular structure, is more stable than linear mRNA and can
prolong protein expression. DNA carriers,
through transposon systems, enable stable integration and sustained expression. Despite
optimization, challenges remain in persistence,
immunogenicity, and editing efficiency. Overall, nanoparticle platforms offer a flexible,
safe, and scalable option for in vivo CAR-T therapy,
representing a promising direction for future immune cell therapies.
In Vivo CAR-T Generation Based on Implant
Beyond direct injection of viruses or nanoparticles, scaffold implants have emerged as a
promising strategy for localized in vivo CAR-T delivery. Scaffold-mediated in vivo CAR-T
generation is primarily achieved in two approaches.
The first approach pre-isolates T cells from patient blood, co-encapsulates them with a CAR
vector in a scaffold, and implants it at the tumor site for in vivo CAR-T generation.
Although it requires brief ex vivo isolation, T cell activation and expansion occur entirely
in vivo.
The second approach directly implants a scaffold with the CAR vector at the disease site to
recruit and edit T cells in vivo. For example, Inamdar et al. used a porous collagen implant
to recruit immune cells. The vector assembles CAR molecules, followed by rapid T cell
expansion and release into tumors, suppressing growth and prolonging survival in mice[4]. Compared to direct vector injection, implants enable rapid
local CAR-T generation and reduce off-target expression.
In summary, implants provide an in situ platform for CAR integration, T cell expansion, and
activation. This approach addresses limitations in CAR-T numbers and enables sustained local release. Compared
to systemic approaches, it offers better efficacy and safety and could become a versatile
platform for CAR-T engineering.
Clinical Progress and Ongoing Challenges of In Vivo CAR-T Therapy
Clinical Progress of in Vivo CAR-T Therapy
Leveraging these diverse viral and non-viral platforms, several in vivo CAR-T candidates
have progressed into clinical trials (Table 3), representing a critical step toward
translating this technology into tangible therapies. The therapeutic applications of in vivo
CAR-T platforms vary according to the delivery technology: viral vectors are primarily
focused on hematological malignancies and autoimmune disorders, whereas non-viral vectors
demonstrate broader applicability, encompassing autoimmune diseases, solid tumors, and
fibrotic conditions (Figure 3).
Figure 3. The clinical landscape of in vivo CAR-T based on viral and non-viral systems[1].
Table 3. Representative in vivo CAR cell therapies advancing to clinical trials[1].
| Products |
Delivery vector |
T cell-specific antigen |
CAR construct |
Indication |
| INT2104 |
LV |
CD7 |
CD20 scFv/4-BB |
R/R B-cell malignancies |
| UB-VV111 |
LV |
CD3 |
CD19 scFv/4-BB |
R/R large B-cell lymphoma and chronic lymphocytic leukemia |
| ESO-T01 |
LV |
TCR |
BCMA VHH/4-1BB |
R/R multiple myeloma |
| KLN-1010 |
LV |
CD3 |
Fully human BCMA CAR |
R/R multiple myeloma |
| OriV508 |
LV |
CD3, CD7 |
Loop CD19 and BCMA CAR |
R/R B-cell malignancies |
| LVIVO-TaVec100 |
LV |
CD3 |
Bicistronic CD20 and CD19 CAR |
R/R B-cell malignancies |
| CPTX2309 |
LNP |
CD8 |
CD19 scFv/CD28 |
R/R autoimmune diseases |
| MT-302 |
LNP |
FcRγ |
TROP2 scFv/CD89
(mRNA) |
Advanced epithelial tumors |
| MT-303 |
LNP |
FcRγ |
GPC3 scFv/CD89 (mRNA) |
Advanced hepatocellular carcinoma |
LNP: lipid nanoparticle, LV: lentiviral vector, TROP2: trophoblast cell surface antigen 2,
GPC3: glypican-3, BCMA: B-cell maturation antigen, VHH: variable domain of heavy chain of
heavy-chain antibody, R/R: relapsed or refractory.
These early clinical studies highlight the translational potential of in vivo CAR-T therapy,
demonstrating encouraging progress across multiple disease indications.
The Potential Challenges of In Vivo CAR-T Manufacturing
While in vivo CAR-T therapy holds great promise, realizing its full clinical potential
requires overcoming key manufacturing- and delivery-related challenges (Figure 4).
Figure 4. Potential risks and related barriers to the realization of in vivo CAR-T
therapy[2].
1. The potential risks of CAR gene delivery
A key step in in vivo CAR-T therapy is the targeted delivery of the CAR gene to T cells.
However, there is a risk of off-target delivery, where the CAR gene may enter non-target
cells, causing antigen masking and preventing CAR-T cells from recognizing and killing tumor
cells. To mitigate this risk, researchers are developing T-cell-specific promoters and other
precise delivery strategies to achieve functional CAR expression within T cells.
2. CAR expression efficiency and regulation
The efficiency of CAR gene expression is a critical determinant of therapeutic outcomes.
Although viral vectors enable stable genomic integration, their transduction efficiency in
primary T cells remains suboptimal. Non-viral vectors, such as nanoparticles, allow for more
controllable CAR expression to reduce toxicity; however, delivery efficiency is often low
and expression is transient, frequently requiring repeated administration. Emerging
technologies, including self-amplifying mRNA (saRNA) and synthetic biology-based tunable
promoter systems, offer promising solutions: they enable highly efficient and sustained CAR
expression even at low doses, helping to lower production costs, reduce carrier payload, and
better balance efficacy with safety.
3. Immunogenicity
In vivo CAR-T therapy may provoke immune responses due to non-self components of the CAR
protein, residual carrier proteins, mRNA constructs, and nanocarriers, potentially affecting
therapeutic efficacy. Strategies to reduce immunogenicity include using humanized CARs,
developing low-immunogenicity carriers, chemically modifying RNA, and optimizing the LNP
delivery system. These approaches can lower the risk of immune clearance, thereby improving
treatment safety and efficacy.
4. Fratricide
CAR-T cells may attack each other by recognizing target antigens expressed on their own or
neighboring T-cell surfaces, a phenomenon particularly observed in therapies targeting
T-cell malignancies. Trogocytosis can also lead to antigen transfer, triggering CAR-T cell
exhaustion. Strategies to mitigate these effects include dynamic regulation of CAR
expression, selecting lower-affinity CARs, and targeting key regulatory molecules (e.g.,
ATF3/
CH25H) to reduce trogocytosis and sustain antitumor
activity.
Summary
Since the first approval in 2017, CAR-T therapy has revolutionized cancer treatment,
particularly for hematological malignancies, and has shown promising potential in
non-oncological conditions such as autoimmune disorders. To overcome the manufacturing
complexities, supply limitations, and high costs associated with conventional adoptive cell
therapy, in vivo CAR-T has emerged as a transformative paradigm. By enabling direct, in situ
generation of CAR-equipped immune cells, it offers a more efficient, accessible, and potentially
"off-the-shelf" therapeutic approach. As the field continues to evolve, in vivo CAR-T is poised
to reshape the landscape of cell-based immunotherapy. Nevertheless, realizing its full potential
requires careful management of persistent challenges related to targeted delivery, precise CAR
expression control, immunogenicity, and long-term safety.
Recommended LNP Lipid Products
Recommended Targeted Delivery LNP Products
| Product Name |
Cat. No. |
Characteristic |
| DSPE-PEG-Maleimide ammonium, MW 3400 |
HY-W441009A |
-Mal can be used for subsequent antibody conjugation |
| DSPE-PEG-Folate, MW 3350 |
HY-144009 |
Folate has a targeting effect and can bind to folate receptors in cancer cells |
| Fluorescent DOTAP |
HY-143702 |
Fluorescence modification can be used for tracking nanocarriers |
| DSPE-PEG2000-iRGD |
HY-172494 |
iRGD can combine with AV-integrins to achieve the effect of tumor penetration |
| DSPE-PEG2000-T7 |
HY-172723 |
T7 specifically binds to TfR |
| DSPE-PEG2000-cRGD |
HY-172464 |
cRGD peptide can specifically bind to αvβ3 on the surface of many cancer cells and
neovascular cells |
| DSPE-PEG2000-GE11 |
HY-172470 |
GE11 can be used for cancer cells with EGFR overexpression |
Note: MCE can provide products for research use only. We do not sell to patients.
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Overview of In Vivo CAR-T Therapy
Platforms and Strategies for In Vivo CAR-T Cell
Production
Clinical Progress and Ongoing Challenges of In Vivo
CAR-T Therapy
