In Vivo CAR-T Integration Site Detection

In Vivo CAR-T Integration Site Detection

1. Project Background

In vivo CAR-T therapy has replaced the traditional in vitro modification and culture mode for cells and their introduction into the patient. By directly injecting viral vectors carrying tumor antigen receptor-encoding genes into the patient, the patient's own in situ T cells are transformed into "universal CAR-T cells" through the "directed drug delivery" method. This innovative technology has greatly enhanced the therapeutic feasibility and efficiency. However, it also introduces safety assessment challenges, especially concerns about long-term adverse effects caused by integration.

The main difference between in vivo CAR-T therapy and traditional ex vivo methods lies in the fact that in vivo CAR-T therapy undergoes integration in multiple tissue organs throughout the body, rather than in a single, controllable cell population. Unlike traditional integration site analysis (Integration Site Analysis, ISA), which relies on peripheral blood mononuclear cells (PBMC) samples, this method is limited to a few "monitoring areas" in the in vivo setting. Since the vector can integrate into various tissue organs such as the lymph nodes, spleen, and liver, causing unpredictable transduction and integration, it is difficult to comprehensively assess integration risk only through circulating cells, and it is even more difficult to detect the occurrence of off-target effects in solid tissue organs.

Cell-free DNA (cfDNA) liquid biopsy technology provides an ideal solution to this critical issue. cfDNA is primarily DNA fragments released into the bloodstream from cells in all tissues and organs of the body (including those undergoing damage or repair), and it is a "molecular barcode" that reflects gene alteration events throughout the body. By performing highly sensitive and broad-spectrum detection of gene integration points in cfDNA through a base-vector junction-based method, comprehensive, dynamic monitoring of the entire integration map and tumor spectrum of in vivo CAR-T can be achieved without invasive procedures, enabling early prediction of potential risks before they emerge in the circulating cells.

Figure 1: Principle of In Vivo CAR-T Whole-Body Integration Site Dynamic Monitoring Based on Plasma cfDNA Liquid Biopsy

2. Project Principle

The core technology of this project is based on linker-mediated PCR (Linker-Mediated PCR, LM-PCR) combined with "expansion-amplification" technology and unique molecular identifier (UMI) technology to achieve high-sensitivity and high-specificity detection of viral integration sites in cfDNA. This technology is called LiBIS-seq (Liquid Biopsy Integration Site Sequencing) [2].

The entire detection process consists of the following key steps:

(1) cfDNA Extraction: High-quality cfDNA is extracted from peripheral blood plasma samples. cfDNA mainly comes from whole-body tissues and organs or dying cells, with an average fragment length of approximately 167bp.

(2) Library Construction and UMI Labeling: The extracted cfDNA undergoes end-repair, addition, and subsequent ligation steps containing UMI detection sequences. UMI technology adds a unique "barcode" to each original DNA molecule, which can be used to remove PCR duplication bias and sequence errors during post-analysis data processing, significantly eliminating PCR expansion bias and sequencing errors.

(3) Linker Connection: Bi-directional linkers (Linker cassette) are connected to the ends of cfDNA fragments, laying the foundation for subsequent PCR expansion and extraction of integration sites.

(4) LM-PCR Amplification: Using primers specific to chronic carrier LTR (long terminal repeat sequence) and the linker for nesting and nested PCR amplification, specifically amplifying DNA fragments containing the base-vector junction point.

(5) NGS Sequencing and Analysis: High-depth sequencing of amplification products (typically ≥50,000X), followed by bioinformatics analysis of the generated information to achieve integration site identification, quantification, and risk assessment.

Figure 2: LiBIS-seq Integration Site Detection Library Construction Flowchart

3. Technical Advantages

*Scope of Technology Application: This technology is designed for in vivo gene therapy scenarios and is also applicable to safety monitoring of ex vivo CAR-T therapy methods, providing important supplementation to traditional PBMC testing.

4. Application Scenarios

4.1 Non-Clinical Research Phase (Animal Models)

(1) Biodistribution and Tropism Confirmation: In large animal models such as NHP, cfDNA analysis can be performed without tissue organ dissection to verify the contribution of viral vector delivery to the tropism characteristics.

(2) Long-term Safety Assessment: During long-term management research, dynamic monitoring of integration site depletion is performed to assess the risk of potential pathogenesis, providing key safety data for clinical trials.

(3) Off-target Effect Evaluation: Monitor the integration of vector in non-target tissues (such as liver, spleen) to evaluate the specificity of in vivo delivery.

4.2 Clinical Research Phase (Human Trials)

(1) Personalized Risk Baseline Establishment: Establish a comprehensive personalized integration site risk map for each participant after the first administration to identify high-risk integration events.

(2) Clonal Dynamic Long-term Monitoring: Perform cfDNA detection regularly during long-term follow-up (LTFU) periods of 15 years, dynamically tracking clonal depletion and degree changes, and detecting abnormal expansion signals in a timely manner.

(3) Safety Event Return: If rare tumor progression events occur that are not well-characterized, this technology can be used to quickly determine the integration site at risk for pathogenesis, providing decisive evidence for event return.

(4) Regulatory Reporting Support: Provide integration site safety data that meets FDA/NMPA requirements for IND/BLA reporting.

5. Case Analysis

To demonstrate the practical application value of this technology in CAR-T cell therapy safety monitoring, we present a case of anti-CD19 CAR-T cell therapy for B-cell lymphoma as an example, showing the integration site dynamic monitoring results from serial therapy at 12 months.

5.1 Integration Site Depletion Trajectory Analysis

Through patient-specific time-point plasma cfDNA LiBIS-seq analysis, we successfully identified and tracked 45 independent chronic viral integration sites. The following are the dynamic changes of 6 major clones (abundance >5%) within 12 months of follow-up:

(1) IS-001 (PTEN gene intron): Baseline abundance 25.3%, showing a slow decline trend during the follow-up period, down to 19.2% at 12 months. PTEN is an important tumor suppressor gene, and this integration site deserves continued attention, but no abnormal expansion has been observed so far.

(2) IS-002 (TP53 gene upstream 50kb): Baseline abundance 18.7%, showing slow upward trend during the follow-up period, up to 23.5% at 12 months. Although TP53 is a classic tumor suppressor gene, this integration site is quite distant from the gene, targeting is relatively low, and the degree of expansion is slow, belonging to normal range wave motion.

(3) IS-003 (KRAS gene downstream 20kb): Baseline abundance 15.2%, maintaining relative stability during the follow-up period, down to 13.1% at 12 months. KRAS is a proto-oncogene, but the integration site is outside the gene, with relatively low risk.

(4) IS-004 (MYC gene upstream 100kb): Baseline abundance 12.4%, showing slow decline trend during the follow-up period, down to 9.8% at 12 months.

(5) IS-005 (BRCA1 gene intron): Baseline abundance 8.9%, maintaining stability during the follow-up period, at 7.9% at 12 months.

(6) Other clones: Total proportion 19.5%-26.5%, consisting of multiple low-to-moderate abundance clones (<5%), showing multiple low-abundance additions, demonstrating the diversified nature of CAR-T cell populations.

Key Conclusion: During the 12-month follow-up period, no sharp expansion of any single clonal group (defined as a more than 10-fold increase in relative abundance) was observed, indicating that the patient's CAR-T cell population maintained multi-clonal nature without obvious clone dominance and safety is good.

Table 1: Main Integration Site Detection Results (Top 10)

Figure 3: Clonal Dynamic Tracking (12-month Follow-up)

Figure 4: Genomic Distribution of Integration Sites (Left) and Risk Classification of Integration Sites (Right)

5.2 Integration Site Gene Group Distribution and Risk Assessment

Analysis of the gene group characteristics of 45 integration sites shows:

(1) 68.5% of integration events occur in the gene intron (mainly within the gene sequence), which is consistent with known characteristic knowledge reported in the active region literature of viral vector bias integration [2].

(2) 12.3% are located in the activating gene sequence, and these integration sites may influence gene expression, requiring key attention.

(3) 8.7% are located in enhancer regions, which may influence gene expression through long-range regulation.

(4) 7.2% are located in gene intervals, which are generally considered relatively low-risk.

(5) 3.3% are located in repeat sequences, the integration will fail to identify the event in a precise manner, but the example is relatively low.

Risk Assessment:

(1) Among 45 integration sites, there are 2 (4.4%) integrations detected near known cancer genes (KRAS and MYC), but they are located at a relatively far distance from the base gene, and no abnormal expansion has been observed so far.

(2) There is 1 (2.2%) integration detected in the tumor suppressor gene intron (PTEN), which has been decreasing gradually over the follow-up period, without abnormality.

(3) Overall Risk Assessment: Low. The integration sites of the majority are distributed within the anticipated range, showing no high-risk integration events detected in the follow-up period. The clonal dynamics are stable, and it is recommended to continue regular monitoring for 3-6 months.

6. Service Content

*Detection Cycle: Approximately 30 working days after sample reception

7. Sample Requirements

To ensure the accuracy and reliability of detection results, please strictly follow the sample requirements below:

(1) Sample Type: Peripheral blood;

(2) Collection Tube: It is recommended to use cfDNA-specific storage tubes (such as Streck tubes) to prevent DNA release from white blood cell lysis into the base genome;

(3) Blood Volume Requirements: 8-10 mL peripheral blood to ensure sufficient high-quality blood (≥4 mL);

(4) Processing Time Limit: Blood samples should be separated within the specified time (subject to tube instructions) for blood separation;

(5) Storage and Transport: Blood samples should be stored frozen at -80°C and shipped on dry ice.

8. References

[1] Biffi, A. Montini, E. Lorioli, L. Cesani, M. Fumagalli, F. Plati, T. ... & Naldini, L. (2013). Lentiviral hematopoietic stem cell gene therapy benefits metachromatic leukodystrophy. Science, 341(6148), 1233158.

[2] Cesana, D. Calabria, A. Rudilosso, L. Gallina, P. Benedicenti, F. Spinozzi, G. ... & Montini, E. (2021). Retrieval of vector integration sites from cell-free DNA. Nature Medicine, 27(7), 1296-1304.