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In Vivo CAR-T Integration Site Detection Solution

In Vivo CAR-T Integration Site Detection Solution

ISA
FDA 15-Year LTFU Compliant · NMPA Latest Guidelines

In Vivo CAR-T
Integration Site Detection
Non-Invasive Whole-Body Monitoring via cfDNA

In vivo CAR-T transforms "personalized cell manufacturing" into "off-the-shelf gene medicine", eliminating leukapheresis and preconditioning chemotherapy. However, random vector integration poses oncogenic risks. Traditional PBMC-based assays fail to detect integration events in solid tissues, creating critical safety blind spots.

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Complete Regulatory to Solution Logic
01
Regulatory Mandate15-year long-term follow-up required for integration safety
02
Traditional LimitationPBMC only detects circulating cells, misses tissue reservoirs
03
LiBIS-seq SolutioncfDNA whole-body monitoring with 0.01% sensitivity
✓ GMP Grade Laboratory · Fully Traceable Data
✓ 40+ Core Patents · Proprietary Assays
✓ 6 ASGCT 2026 Abstracts Accepted
✓ Served 50+ Global CGT Programs
INDUSTRY BACKGROUND

The Next Revolution in Cell Therapy

In vivo CAR-T is entering clinical validation at an unprecedented pace. Major pharmaceutical companies are investing billions in acquisitions, but integration-related oncogenic risk remains the biggest bottleneck to widespread adoption.

Integrating Vector Technology

Engineered lentiviral (eLV) and γ-retroviral vectors achieve targeted T cell transduction in vivo, providing long-term therapeutic efficacy. However, semi-random integration can activate proto-oncogenes or disrupt tumor suppressor genes, leading to clonal expansion and secondary malignancies. Long-term dynamic monitoring of integration sites is therefore a mandatory safety requirement.

Non-Integrating Vector Technology

Lentiviral nanoparticles (LvNP) eliminate integrase elements, theoretically preventing genomic integration. However, this "non-integrating" safety claim requires rigorous experimental validation. Integration site analysis remains critical for LvNP products to confirm the absence of unintended integration events for IND/BLA submissions.

Critical Industry Pain Point: The Monitoring Blind Spot

Unlike ex vivo CAR-T where gene modification occurs in a controlled cell population, in vivo therapy transduces cells across multiple organs. Traditional integration site analysis relies solely on peripheral blood mononuclear cells (PBMC), which cannot capture integration events in lymph nodes, spleen, liver, or other solid tissues. This creates a dangerous blind spot where abnormal clones can expand undetected for months or years.

TECHNICAL PRINCIPLE

LiBIS-seq: The Gold Standard for cfDNA ISA

Our proprietary Linker-Mediated PCR with Unique Molecular Identifiers (UMI) enables highly sensitive and specific detection of lentiviral integration sites in cell-free DNA, providing a molecular snapshot of genomic events across the entire body.

1

cfDNA Extraction

High-quality cfDNA is isolated from peripheral blood plasma samples. cfDNA primarily originates from apoptotic or necrotic cells throughout the body, with an average fragment length of approximately 167 bp.

2

Library Construction & UMI Labeling

Extracted cfDNA undergoes end repair and A-tailing, followed by ligation of sequencing adapters containing UMIs. UMI technology assigns each original DNA molecule a unique "barcode", effectively eliminating PCR amplification bias and sequencing errors.

3

Linker Ligation

A partially double-stranded linker cassette is ligated to the ends of cfDNA fragments, providing specific primer binding sites for subsequent PCR amplification.

4

LM-PCR Amplification

Two rounds of nested PCR using lentiviral LTR-specific primers and linker-specific primers are performed to specifically enrich DNA fragments containing virus-host genome junction sequences.

5

NGS Sequencing & Analysis

Amplification products are subjected to ultra-high-depth sequencing (typically ≥50,000x). Bioinformatics analysis is then applied for integration site identification, quantification, and risk assessment.

Detection Sensitivity: 0.01%
CORE ADVANTAGES

Four Defining Technical Advantages

Purpose-built for in vivo gene therapy scenarios, addressing all limitations of traditional integration site assays and meeting global regulatory requirements.

Comprehensive Representativeness

Overcomes the "monitoring blind spot" of conventional PBMC assays by capturing integration signals from all solid tissues including lymph nodes, spleen, and liver, providing a panoramic view of systemic integration risk critical for in vivo therapies.

Ultra-High Sensitivity

Combining UMI error correction and nested LM-PCR enrichment, achieves a detection limit of 0.01%, enabling precise identification and quantification of rare abnormal clones hidden within vast amounts of background DNA.

Non-Invasive Dynamic Monitoring

Requires only 8-10 mL of peripheral blood, no tissue biopsies needed. Enables high-frequency, continuous monitoring of clonal evolution over time, fully complying with FDA's 15-year long-term follow-up (LTFU) requirements.

Early Warning Capability

Detects potential dominant clonal expansion earlier than clinical symptoms or conventional hematological indicators, providing a critical time window for clinical intervention and risk management.

APPLICATIONS

Full Lifecycle Coverage

Supports all stages from preclinical research to commercialization, providing critical safety data to accelerate regulatory approval and ensure patient safety.

NON-CLINICAL RESEARCH

Animal Models & IND Submission

Core Applications
Biodistribution & Targeting ValidationLarge Animal Long-Term ToxicologyOff-Tissue Transduction AssessmentTumorigenicity Risk EvaluationIND Submission Data Package
CLINICAL DEVELOPMENT

Clinical Trials & Commercialization

Core Applications
Individualized Risk Baseline Establishment15-Year Long-Term Clonal MonitoringSerious Adverse Event AttributionBLA Submission Data PackagePost-Marketing Safety Surveillance
CASE STUDY

12-Month Integration Site Monitoring

A B-cell lymphoma patient treated with anti-CD19 CAR-T therapy underwent comprehensive integration site monitoring via LiBIS-seq, demonstrating an excellent safety profile.

45
Unique Integration Sites
Successfully identified and tracked all major clones
62.2%
Low-Risk Sites
28 sites located in genomic safe regions
0
Abnormal Clonal Expansion
No single clone increased >10-fold in abundance

Key Clinical Findings

  • Stable Clonal Dynamics: All 6 major clones (abundance >5%) showed gradual fluctuations without any signs of rapid expansion
  • Expected Genomic Distribution: 64.4% of integrations occurred within genes (predominantly introns), 15.6% in promoter regions, and 11.1% in enhancers, consistent with known lentiviral integration preferences
  • Low Overall Risk: Only 3 sites (6.7%) were near known oncogenes but at significant distances, and 1 site within a tumor suppressor gene showed declining abundance

Conclusion: The patient maintained a polyclonal CAR-T cell population with no evidence of high-risk integration events. Continued monitoring every 3-6 months is recommended.

SERVICE OFFERING

Standardized End-to-End Service

Full-process standardized service from study design to report delivery, strictly following GMP guidelines to ensure data quality and on-time delivery.

Service Scope & Deliverables

  • Collaborative design of integration site monitoring protocols aligned with FDA/NMPA requirements
  • Standardized guidelines for plasma collection, processing, and shipping
  • High-quality cfDNA extraction and rigorous quality control
  • LiBIS-seq ultra-deep sequencing and specialized bioinformatics analysis
  • Comprehensive report including integration site list, clonal dynamics, genomic distribution, and risk assessment with expert interpretation

Standard Turnaround Time: 30 business days from sample receipt

Sample Requirements

  • Sample Type: Peripheral blood plasma
  • Collection Tube: cfDNA-specific preservation tubes (e.g., Streck tubes) recommended to prevent leukocyte lysis
  • Volume Requirement: 8-10 mL peripheral blood (≥4 mL plasma)
  • Processing Time: Separate plasma promptly according to collection tube instructions
  • Storage & Shipping: Store plasma at -80°C, ship on dry ice, avoid repeated freeze-thaw cycles
GET STARTED

Developing an In Vivo CAR-T Program?

Tell us about your product type, technology platform, and development stage. We will provide a customized integration site monitoring plan and timeline compliant with FDA/NMPA requirements.

Call Us +86 153 3655 7985 →Download Technical Whitepaper
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