A Listeriolysin O antibody-toxin conjugate (ATC) targets CD47 for cancer immunotherapy
An engineered antibody-toxin conjugate targeting CD47, linked to listeriolysin O, signals immune cells to destroy the targeted tumor cell. Schrank et al. (2025) recently engineered an antibody-toxin conjugate targeting the immune system's "don't eat me" signal, CD47. The antibody in this ATC is linked to the bacterial toxin listeriolysin O from the intracellular bacterium Listeria monocytogenes (Lm) via a cleavable linker (CD47-LLO).
CD47 Antibody-LLO Toxin Conjugate
To create the ATC, the researchers conjugated the CD47 antibody to Listeriolysin O (LLO) using the click-chemistry labeling reagent DBCO-PEG4-NHS ester and the crosslinking reagent SPDP-PEG11-azide. Under reducing conditions, disulfide bonds are reduced, and LLO dissociates from the conjugate, exerting its beta-hemolytic activity.
Listeriolysin O (LLO) contains a single, highly conserved reactive cysteine residue (Cys484) located within its C-terminal undecapeptide domain, situated in the conserved 11-amino-acid signature motif (ECTGLAWEWWR) shared across cholesterol-dependent cytolysins (CDCs). Cys484 acts as a redox-sensitive switch that modulates pore-forming and hemolytic activity. However, the free thiol group makes the toxin vulnerable to oxidative inactivation and S-glutathionylation, but the addition of reducing agents can fully restore its lost hemolytic activity.
Host factors like the γ-Interferon-inducible lysosomal thiol reductase (GILT) target this cysteine inside the phagosome. The redox interaction controls or triggers LLO activity specifically where the bacterium needs to escape into the host cell cytoplasm.
CD47-LLO delivered directly to the mouse tumor produced a systemic antitumor response, bridging innate and adaptive immunity. Systemic delivery of CD47-LLO also resulted in broader immune activation. Cytokine levels in animal serum increased hours after intraperitoneal administration (injection into the fluid-filled space inside the abdomen), and an anti-drug-antibody response arose weeks after the animal’s treatment was completed.
CD47
CD47 is a cell-surface protein that acts as an innate immune checkpoint and a "don't eat me" signal to prevent myeloid cells like macrophages from engulfing healthy or cancerous cells. CD47 binds to SIRPα (Signal Regulatory Protein Alpha) on myeloid cells, including macrophages and dendritic cells, recruiting tyrosine phosphatases (SHP-1/SHP-2), which stop myosin accumulation at the synapse and suppress cell clearance. Normal cells use this pathway to signal that they belong in the body and should not be destroyed. Many tumors, for example, myeloid cancers like Acute Myeloid Leukemia (AML) and Myelodysplastic Syndrome (MDS), aberrantly overexpress CD47 to hide from the immune system. High CD47 expression often correlates with disease progression and poor patient survival.
Blocking antibodies or decoy receptors disrupt the CD47-SIRPα interaction. Inhibiting this checkpoint targets macrophages to phagocytose (engulf) and eliminate tumor cells. Because CD47 is widely expressed on normal red blood cells, early-generation therapies sometimes faced side effects like anemia from red blood cell clearance, driving the development of safer engineered decoys and dual-targeting agents.
Listeriolysin O
Listeriolysin O (LLO) is a cholesterol-dependent pore-forming toxin produced by Listeria monocytogenes that primarily enables the bacterium to escape from the host phagosome into the cytosol. This Gram-positive foodborne facultative intracellular bacterial pathogen can cause serious foodborne infections in immunocompromised individuals and pregnant women.
LLO targets and binds to cholesterol molecules present in host cell membranes. After binding, LLO monomers assemble into arc-shaped or ring-like oligomers, creating structural perforations and membrane defects that disrupt the bilayer. LLO's acid-pH optimum of around pH 5.5 to 6.0 matches the intracellular phagosome environment, ensuring the toxin punches holes in the vacuolar membrane while remaining largely inactive at the neutral cytosolic pH of 7.2, protecting the host cell's outer plasma membrane from premature lysis. LLO contains a unique N-terminal PEST-like sequence that targets cytosolic LLO for rapid degradation or triggers host AP-2-dependent endocytosis, removing misdirected toxin from the plasma membrane and preserving cell integrity.
Chen et al. discovered that the PEST-like sequence of LLO interacts with a cellular endocytosis adaptor to promote endocytosis of plasma membrane-associated LLO, preventing its cytotoxicity to the infected cell and enabling Listeria pathogenesis.
By compromising the phagosomal membrane without destroying the entire infected cell, LLO allows Listeria to enter the cytoplasm, replicate, and spread to neighboring cells.
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