Development of a Novel HER2-CAR Monocyte Cell Therapy with Controllable Proliferation and Enhanced Anti-Tumor Efficacy
Introduction
Recent advancements in cellular immunotherapies, particularly chimeric antigen receptor (CAR)-T cell therapies, have shown promise in treating hematological malignancies. However, these therapies face significant challenges in treating solid tumors due to the dense tumor matrix, which hampers CAR-T cell infiltration, and the immunosuppressive tumor microenvironment (TME) that weakens their function. Additionally, uncontrolled toxicities such as cytokine release syndrome (CRS) and cerebral neurotoxicity pose risks. To overcome these obstacles, novel CAR cell strategies are urgently needed.
Macrophages, which are frequently enriched within the TME, offer a promising alternative. Intravenously injected macrophages can infiltrate the interior portion of the tumor, making CAR-monocytes/macrophages (CAR-M) a viable therapeutic option. However, autologous macrophage infusion has shown limited anti-tumor efficacy, likely due to M2 polarization in the immunosuppressive TME. Genetic engineering of monocytes as CARs against tumor cells is a novel strategy to address this limitation.
This study aimed to develop a novel CAR-M based on the THP1 immortalized human monocyte cell line, incorporating the suicide gene inducible caspase-9 (iCasp9) and anti-erb-b2 receptor tyrosine kinase 2 (HER2) CAR elements. The goal was to create a therapy with controllable proliferation, enhanced anti-tumor efficacy, and the ability to bridge innate and adaptive immunity.
Methods
The study involved several key steps to construct and evaluate the novel CAR-M. The iCasp9 and HER2-CAR elements were transfected into THP1 cells using lentivirus. The suicide efficiency and specific anti-tumor efficacy were assessed using flow cytometry, inCucyte, and tumor-bearing mouse models. Transcriptome sequencing was performed to explore the activation of related signaling pathways in CAR-THP1 cells. Finally, the synergistic therapeutic efficacy of CAR-THP1 combined with Retronectin activated killer cells (RAKs) was demonstrated in tumor-bearing mouse models.
Results
The iCasp9 gene endowed monocyte cell lines with controllable proliferation. THP1 cells stably expressing iCasp9 (iCasp9-THP1) exhibited over 99% expression efficiency and remained stable for up to 30 days. The iCasp9-THP1 cells initiated suicide quickly and efficiently under the control of the iCasp9 gene, with complete cell death observed within 14 days post-treatment with 20 nmol/L AP1903. In vivo studies confirmed the rapid elimination of iCasp9-THP1 cells in the abdominal cavity and peripheral blood following AP1903 administration.
The HER2-CAR-CD3ζ-CD147 permutation was identified as the optimal combination for CAR-THP1. This configuration exhibited the highest specific phagocytosis efficiency and cytotoxicity against HER2-positive ovarian cancer cells (SKOV3). CAR-THP1 also demonstrated significantly elevated metalloproteinase-13 (MMP13) secretion, which facilitated the degradation of the tumor matrix and enhanced immune cell intratumoral infiltration.
In tumor-bearing mouse models, CAR-THP1 treatment significantly inhibited tumor growth and increased the number of apoptotic tumor cells. Multiplex immunofluorescence staining revealed elevated CD86 expression levels and reduced CD206 expression levels in CAR-THP1 cells, indicating a shift toward the M1 phenotype. Transcriptome sequencing further confirmed that CAR-THP1 activation triggered additional signaling pathways, promoting the secretion of pro-inflammatory cytokines and enhancing the activation of natural killer (NK) cells, NKT cells, and T cells.
The combination of CAR-THP1 with RAK immunotherapy showed superior therapeutic efficacy compared to CAR-THP1 treatment alone. CAR-THP1 facilitated the intratumoral infiltration of RAK cells, leading to a significant reduction in tumor size and increased apoptosis. This synergistic effect was attributed to the MMPs secreted by CAR-THP1, which degraded the dense tumor matrix and enhanced the anti-tumor immune response.
Discussion
The development of CAR-THP1 represents a significant advancement in CAR-M therapy. The incorporation of the iCasp9 suicide gene addressed concerns regarding uncontrolled proliferation in vivo, providing a safety mechanism to eliminate residual CAR-THP1 cells post-treatment. The use of the THP1 immortalized human monocyte cell line as the cell source for CAR-M overcame the limitations of autologous monocytes, offering a sustainable and reliable option for long-term culture and preservation.
The HER2-CAR-CD3ζ-CD147 permutation demonstrated superior specific phagocytosis efficiency and cytotoxicity against HER2-positive tumor cells. The inclusion of the CD147 intracellular segment promoted MMP13 secretion, facilitating immune cell intratumoral infiltration and enhancing the anti-tumor immune response. Transcriptome sequencing revealed that CAR-THP1 activation triggered additional signaling pathways, promoting the secretion of pro-inflammatory cytokines and enhancing the activation of NK cells, NKT cells, and T cells.
The combination of CAR-THP1 with RAK immunotherapy showed superior therapeutic efficacy, highlighting the potential of CAR-THP1 to bridge innate and adaptive immunity. This synergistic effect was attributed to the MMPs secreted by CAR-THP1, which degraded the dense tumor matrix and enhanced the intratumoral infiltration of RAK cells.
Conclusion
CAR-THP1 represents a promising therapeutic option for HER2-positive tumors, with the potential for combination therapy with other immune cells. The incorporation of the iCasp9 suicide gene, the use of the THP1 immortalized human monocyte cell line, and the HER2-CAR-CD3ζ-CD147 permutation collectively address the challenges of CAR-M therapy, offering a safe, effective, and sustainable treatment option.
doi.org/10.1097/CM9.0000000000002944
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