AAV-Mediated Trastuzumab Delivery to the Central Nervous System for EGFR2-Positive Brain Metastases

Introduction

In the treatment of breast cancer, tumors that are human epidermal growth factor receptor 2 (HER2) positive exhibit more aggressive characteristics, posing significant challenges for clinical treatment. Since the approval of trastuzumab (brand name Herceptin®) in 1998, the drug has significantly improved the overall survival rate of patients with HER2+ breast cancer. However, for HER2+ brain metastases that progress to the central nervous system (CNS), due to factors such as the blood-brain barrier and other influences, trastuzumab has a short half-life in cerebrospinal fluid (2-4 days), rendering traditional systemic anti-HER2 antibody therapies less effective. Therefore, there is a need for new therapeutic approaches targeting HER2+ CNS diseases.

Research Background

This study was conducted by researchers Marcela S. Werner, Shweta Aras, Ashleigh R. Morgan, and others from the Gene Therapy Program at the Perelman School of Medicine, University of Pennsylvania, with results published in the journal Cancer Gene Therapy on March 13, 2024.

Research Details

Research Process

To target HER2+ CNS metastases, the researchers developed a recombinant adeno-associated virus (AAV) vector that expresses a codon-optimized trastuzumab sequence driven by a human ubiquitin C (UBC) promoter (aav9.ubc.trastuzumab). The study included the following main steps: 1. Vector Construction: Optimization of the trastuzumab heavy/light chain sequences, guided by human interleukin 2 (IL2) signal peptide, and separated by furin cleavage and self-cleaving peptide linkers. 2. Animal Model Validation: - Intracerebral injection (ICV) and in vivo anti-tumor experiments using adult RAG1 knockout mice. - Intracisternal magna (ICM) injection using non-human primates (NHPs). 3. Gene Expression and Distribution Detection: - Utilizing real-time quantitative PCR (qPCR), enzyme-linked immunosorbent assay (ELISA), Western Blot, in situ hybridization (ISH), single-cell RNA sequencing (snRNA-seq), and other methods. - Detecting and analyzing anti-tumor effects, with multiple biosample collections for pharmacodynamics evaluation. 4. Anti-Tumor Efficacy Evaluation: - Establishment of breast cancer brain metastasis xenograft models. - Tracking tumor progression through the use of an in vivo imaging system (IVIS).

Research Results

1. Vector Introduction and Expression: After ICV injection of aav9.ubc.trastuzumab (1 × 10^11 gc/mouse), sustained expression of trastuzumab was detected in the serum and brains of RAG1 knockout mice. qPCR data indicated a 1769-fold and 3312-fold increase in vector gene signals in the brain and liver, respectively, with mRNA levels enhanced by 26188-fold and 3967-fold, respectively.
2. Anti-Tumor Effects: aav9.ubc.trastuzumab significantly delayed tumor progression in BT-474 and MDA-MB-453 xenograft models, marked by a significant reduction in tumor-associated optical signals and extended overall survival. For example, in BT-474 xenograft mice treated with the vector, more than half achieved complete remission (>12 weeks).
3. NHP Model Validation: ICM injection (3 × 10^13 gc/subject) in 3-4 year-old female Indian rhesus macaques showed no significant adverse reactions or pathological abnormalities over a period of 36-37 days. ELISA and LC-MS detected sufficient expression of trastuzumab in CSF and brain tissue samples.

Conclusion

This study verified the long-term expression of AAV-mediated trastuzumab in the central nervous system and its significant anti-tumor effects on HER2+ brain metastases. This gene therapy strategy not only avoids complications associated with repeated injections but also improves patient prognosis through sustained local drug concentration.

Research Significance and Highlights

1. Clinical Significance:

   • Provides a new therapeutic approach targeting HER2+ CNS metastases, potentially overcoming the limited efficacy caused by the blood-brain barrier in current treatment regimens.
   • Potentially reduces patient treatment burden and complication risks, significantly improving quality of life.

2. Technological Innovation:

   • Development of a novel AAV vector driven by the UBC promoter and codon-optimized trastuzumab, achieving efficient transduction and long-term expression through ICV and ICM injections.
   • Comprehensive application of three-dimensional imaging technology and real-time quantitative PCR, enhancing data accuracy and reliability.

3. Application of Animal Models:

   • Successful application of NHP models provides robust data support, laying the groundwork for future clinical trials.

Summary

This study fully demonstrates the feasibility and significant anti-tumor effects of using AAV9 vectors to sustain the expression of trastuzumab in the central nervous system. This groundbreaking achievement not only offers new hope for patients with HER2+ CNS metastases but also paves the way for future gene therapy research.