Engineering TME-activated CD47-specific CAR Macrophages

Engineering of TME-activated CD47-specific CAR Macrophages

Authors and Affiliation

  • Fuyu Du, Meixi Jiang, Jingjing Qiu, Anna He, Min Liu, Yuanhui Xu, Xiaocheng Gong, Xinruo Wang, Haotian Zhang, Xianghan Zhang, Xinyi Xu, Lili Lu, Zhongliang Wang, Pengbo Ning

  • Affiliations: School of Life Science and Technology, Engineering Research Center of Molecular & Neuroimaging, College of Veterinary Medicine, School of Medicine and Health, Advanced Diagnostic-Therapy Technology and Equipment Key Laboratory, Clinical Center for Biotherapy.

Citation

Du F, et al. (2025). Engineering TME-activated CD47-specific CAR macrophage via Arg1 promoter for safe and effective solid tumor immunotherapy. Journal of Immunotherapy of Cancer, 13, e012463. doi:10.1136/jitc-2025-012463.

Corresponding Authors

  • Dr. Pengbo Ning: pbning@xidian.edu.cn

  • Zhongliang Wang: wangzl@xidian.edu.cn

Abstract

Background

  • Chimeric antigen receptor macrophage (CAR-Mφ) therapy shows potential in treating solid tumors, but faces issues such as:

    • Target compatibility

    • Systemic toxicity

Methods

  • The study focuses on screening the CD47-scFv sequence in CAR-Mφ as the extracellular framework. Developed a CD47 CAR incorporating a costimulatory domain of α1β1 integrin-mediated Fc-gamma receptor I (FcγRI) signaling.

  • Created a tumor microenvironment (TME)-responsive CAR macrophage platform via Arg1 promoter targeting CD47.

Results

  • Anti-CD47-scFv-mediated macrophages effectively kill tumor cells both in vivo and in vitro.

  • The α1β1 integrin-mediated FcγRI signaling domain enhances the antitumor efficiency of CD47 CAR-Mφ in hCD47+4T1 and SGC-7901 cell models.

  • The Arg1-mediated activation of CD47 CAR-Mφ shows potent cytotoxicity towards cancer cells.

  • Findings illustrate TME-controllable CAR gene expression leading to tumor regression while reducing erythrocyte toxicity.

Conclusions

  • The TME-specific activation mechanism of pArg1 CD47 CAR-Mφ allows safe multidose administration and minimizes systemic toxicity. This creates a Trojan horse-like system for effective solid tumor treatment.

Introduction

  • Cell therapy has shown strong outcomes in hematologic malignancies, but it remains challenging for solid tumors.

  • Various immune cells are being explored for adoptive cell therapy against solid tumors, particularly CAR-Mφ.

  • Studies have shown CD3ζ-based CARs can drive antitumor phagocytic activity.

  • Successful phase I clinical trials by the FDA suggest CAR-Mφ therapy offers a new frontier for solid tumor treatment.

Current Challenges

  • Two main obstacles facing clinical translation of CAR-Mφ:

    • Insufficient cellular activity

    • Off-target toxicity risks, similar to CAR-T therapy

CAR Structure

  • CARs are transmembrane proteins comprising:

    • Antibody single-chain variable fragment (scFv)

    • A hinge region linked to a transmembrane domain with intracellular signaling motifs.

Known Facts About CD47 and Challenges

  • CD47 is a 'do not eat me' signal (overexpressed in many solid tumors) that can limit therapeutic efficacy due to toxicity concerns related to healthy cells. Off-target effects can lead to dose-limiting anemia due to CD47 expression on erythrocytes.

  • There is a need to mitigate CD47 target-mediated toxicity to increase effectiveness without harming healthy cells.

TME Characteristics Influencing Macrophage Behavior

  • The tumor microenvironment fosters a specific metabolic landscape, influencing macrophages to produce arginase 1 (Arg1), which is related to tumor proliferation and associated with poor prognosis in several cancers.

RNA Sequencing Findings

  • RNA sequencing identified significant expression of functional genes following CD47 blockade, revealing pathways linked to:

    • Growth

    • Inflammation

    • Metabolism

  • Changes to CD47-scFv structure show minimal impact on CAR-Mφ efficacy but alpha1beta1-mediated signals enhance anti-tumor activities.

Key Results

Evaluation of CD47-scFvs for CAR Construction

  • Two plasmids with distinct CD47-scFv sequences were constructed and tested for binding affinity to CD47.

    • Binding similarity indicates potential equivalent antitumor activity.

  • Truncated CAR structures were created (CAR-like Mφ) containing:

    • Extracellular CD47-scFv

    • Transmembrane region

    • Intracellular GFP marker.

In vitro Antitumor Efficacy Evaluations

  • CD47-scFv I Mφ achieved 25% phagocytosis of SGC-7901 cells, CD47-scFv II Mφ about 19%, with effective target tumor cell death observed.

In vivo Targeting Studies

  • Using hCD47+4T1 tumor models, CD47-scFv Mφ groups displayed significant tumor targeting and reduction in organ accumulation relative to control groups.

Transcriptomic and Antitumor Analysis

  • Further investigation post-administration of CD47-scFv I Mφ indicated alterations in glycolysis and gluconeogenesis pathways, possibly explaining enhanced tumor suppression.

Construction and Functionality of pArg1 CD47 CAR-Mφ

  • Focused on overcoming erythrocyte toxicity through development of an Arg1 promoter-based CAR-Mφ that remains inactive during circulation but activates in TME.

  • High Arg1 activity correlates with tumor aggressiveness, leading to exploration of the TME for advancements in CAR-Mφ technology.