An Antibody–Toxin Conjugate Targeting CD47 Linked to the Bacterial Toxin Listeriolysin O for Cancer Immunotherapy

Immunology Biochemistry Life Sciences Oncology Medicine
Cancer immunotherapy CD47 Antibody-toxin conjugate Antigen presentation Tumor cell phagocytosis

Academic Background

Cancer immunotherapy has become a hot topic in cancer research in recent years, with its core goal being to activate the patient’s own immune system to recognize and eliminate cancer cells. However, cancer cells evade immune system attacks through various mechanisms, one of which is the expression of the “don’t eat me” signal molecule CD47. CD47 binds to the signal regulatory protein α (SIRPα) on the surface of macrophages, inhibiting the phagocytosis of tumor cells by macrophages, thereby helping tumor cells escape immune surveillance.

Although antibodies targeting CD47 have shown certain therapeutic effects, their use alone is often insufficient to fully activate the immune system. Therefore, researchers have begun exploring ways to further enhance the immune response. This paper proposes an innovative strategy: conjugating the CD47 antibody with the bacterial toxin Listeriolysin O (LLO) to form an antibody-toxin conjugate (ATC), thereby promoting the phagocytosis and antigen presentation of tumor cells and enhancing anti-tumor immune responses.

Source of the Paper

This paper was co-authored by Benjamin R. Schrank, Yifan Wang, and others from multiple institutions, including the University of Texas MD Anderson Cancer Center and the Houston Methodist Research Institute. The paper was published in March 2025 in the journal Nature Cancer, titled “An antibody–toxin conjugate targeting CD47 linked to the bacterial toxin listeriolysin O for cancer immunotherapy.”

Research Process

1. Design and Synthesis of the Antibody-Toxin Conjugate

The researchers first designed the CD47-LLO conjugate. The specific steps are as follows: - Antibody Modification: The CD47 antibody (MIAP410) was modified using the click chemistry reagent DBCO-PEG4-NHS ester. - Toxin Modification: The LLO protein was modified using the cross-linking reagent SPDP-PEG11-azide. - Conjugation Reaction: The modified antibody and LLO were mixed and allowed to react overnight. - Purification: Unconjugated antibodies and LLO were removed via affinity chromatography and size-exclusion chromatography, resulting in the final CD47-LLO conjugate.

2. In Vitro Validation of CD47-LLO Function

The researchers validated the function of CD47-LLO in vitro, including: - Enhanced Phagocytosis: Co-culturing CD47-LLO with macrophages and dendritic cells significantly enhanced the phagocytosis of tumor cells. - Increased Lysosomal Membrane Permeability: Transmission electron microscopy revealed perforations in the lysosomal membranes of macrophages treated with CD47-LLO, indicating that LLO formed pores in the lysosomes, promoting the release of tumor antigens. - Enhanced Antigen Presentation: Macrophages and dendritic cells treated with CD47-LLO showed increased tumor antigen presentation, activating tumor-specific T cells.

3. In Vivo Validation of CD47-LLO Anti-Tumor Effects

The researchers validated the anti-tumor effects of CD47-LLO in multiple mouse tumor models, including: - Breast Cancer Models: In the 4T1BR4 and EO771 breast cancer models, CD47-LLO significantly inhibited primary tumor growth and reduced metastasis. - Melanoma Model: In the D4M.3A melanoma model, CD47-LLO also demonstrated significant anti-tumor effects. - Immune Memory Effect: Long-term surviving mice treated with CD47-LLO exhibited resistance to tumor rechallenge, indicating that CD47-LLO could induce long-lasting anti-tumor immune memory.

4. Mechanism Study

The researchers further explored the mechanism of action of CD47-LLO, finding: - Activation of the cGAS-STING Pathway: CD47-LLO activated the cGAS-STING pathway by releasing tumor DNA, promoting the production of type I interferons. - Synergistic Effects of Macrophages and T Cells: CD47-LLO promoted the polarization of macrophages toward a pro-inflammatory phenotype and increased the infiltration of CD8+ T cells in tumors.

Key Findings

  1. CD47-LLO Significantly Enhances Tumor Cell Phagocytosis: In vitro experiments showed that macrophages and dendritic cells treated with CD47-LLO significantly enhanced the phagocytosis of tumor cells.
  2. CD47-LLO Promotes Tumor Antigen Release and Presentation: Macrophages and dendritic cells treated with CD47-LLO demonstrated increased tumor antigen presentation, activating tumor-specific T cells.
  3. CD47-LLO Shows Significant Anti-Tumor Effects in Multiple Tumor Models: In breast cancer and melanoma models, CD47-LLO significantly inhibited tumor growth and metastasis.
  4. CD47-LLO Activates the cGAS-STING Pathway, Promoting Type I Interferon Production: Mechanistic studies revealed that CD47-LLO activated the cGAS-STING pathway by releasing tumor DNA, further enhancing the anti-tumor immune response.

Conclusions and Significance

This study designed a novel antibody-toxin conjugate, CD47-LLO, which significantly enhances anti-tumor immune responses by promoting tumor cell phagocytosis and antigen presentation. CD47-LLO demonstrated significant anti-tumor effects in multiple tumor models and was able to induce long-lasting anti-tumor immune memory. Additionally, CD47-LLO further enhanced the immune response by activating the cGAS-STING pathway.

This research not only provides a new strategy for cancer immunotherapy but also offers a theoretical basis for developing other antibody-toxin conjugates targeting immune checkpoints. The clinical application prospects of CD47-LLO are broad, and it is expected to become an important tool in cancer immunotherapy.

Research Highlights

  1. Innovative Antibody-Toxin Conjugate Design: CD47-LLO combines the targeting ability of the CD47 antibody with the lysosomal membrane-perforating ability of LLO, significantly enhancing anti-tumor immune responses.
  2. Multi-Model Validation of Anti-Tumor Effects: CD47-LLO demonstrated significant anti-tumor effects in multiple tumor models, including breast cancer and melanoma.
  3. In-Depth Mechanistic Studies: By activating the cGAS-STING pathway, CD47-LLO further enhanced the immune response, providing new insights into its mechanism of action.

Other Valuable Information

This study also found that the combination of CD47-LLO with PD-1 antibodies could further enhance anti-tumor effects, suggesting its potential application in combination immunotherapy. Additionally, systemic administration of CD47-LLO may trigger certain inflammatory responses and anti-drug antibody (ADA) reactions, necessitating further optimization of administration strategies in future clinical applications.