Discover how synthetic Granzyme B molecules are revolutionizing targeted cancer therapy through the innovative COVERT platform
Imagine a cancer treatment so precise it only eliminates cancerous cells while leaving healthy tissue completely untouched. This vision of a "magic bullet" has driven cancer research for decades, yet has remained frustratingly out of reach. Traditional therapies like chemotherapy and radiation often cause severe side effects by indiscriminately targeting both healthy and diseased cells. Even newer immunotherapies, which harness the body's immune system to fight cancer, still face a fundamental limitation: they typically recognize only surface proteins that aren't exclusive to cancer cells, leading to damaging "off-target" effects on healthy tissue1 .
What if we could create a smarter therapeutic that doesn't just stop at the cell surface but actually ventures inside potential target cells to verify their cancerous identity before striking? This is precisely what researchers have achieved with a groundbreaking platform called COVERT (Cytoplasmic Oncoprotein VErifier and Response Trigger), which features a engineered version of a natural killer molecule called Granzyme B1 .
of people will be diagnosed with cancer at some point in their lifetimes
of cancer patients experience severe side effects from traditional treatments
of treatment failures in advanced cancers are due to drug resistance
To appreciate this engineering marvel, we must first understand its foundation. Granzyme B (GrB) is a powerful serine protease—an enzyme that cuts other proteins—produced by our immune system's specialized killers: cytotoxic T-cells and natural killer (NK) cells3 . Its natural role is to eliminate virus-infected and cancerous cells by triggering programmed cell death (apoptosis)2 .
When immune cells identify a threat, they release GrB along with another protein called perforin into the space between cells. GrB then enters target cells and launches a multi-pronged attack on their internal machinery:
Immune cell identifies threat
Releases GrB & perforin
Enters target cell
Triggers apoptosis
This efficient killing machinery makes GrB an attractive candidate for cancer therapy, but with a significant catch: in its natural form, it cannot distinguish between healthy and diseased cells once inside.
The brilliance of the COVERT platform lies in how it transforms this indiscriminate killer into a discerning sleeper agent. Researchers have engineered GrB to remain dormant until activated by specific cancer signals inside target cells1 .
The key insight came from understanding GrB's activation mechanism. Natural GrB is initially produced as an inactive precursor with a small peptide tag that prevents it from functioning. Only when immune cells are ready to deploy their weapons does the enzyme cathepsin C remove this tag, activating GrB by freeing its business end1 3 .
The COVERT platform hijacks this activation system by replacing the natural inhibitory tag with artificial ones that can only be removed by proteases specific to cancer cells. The modular design allows researchers to mix and match different inhibitory tags tailored to recognize various cancer-associated proteases1 .
| Approach | Mechanism | Limitations |
|---|---|---|
| Natural GrB | Directly induces apoptosis in any cell it enters | No discrimination between healthy and diseased cells |
| Antibody-Drug Conjugates | Targets surface proteins on cancer cells | Limited to extracellular targets; off-tumor toxicity |
| COVERT Platform | Requires intracellular cancer proteases for activation | Double verification system significantly improves specificity |
The COVERT platform creates a "double verification" system where both surface recognition AND intracellular cancer signals are required to activate the therapeutic molecule.
In their landmark study, the research team focused on developing a COVERT molecule that would activate in response to SENP1, a protease frequently overexpressed in prostate, pancreatic, and thyroid cancers1 .
The researchers designed a hybrid molecule by fusing the first 97 amino acids of SUMO1 (a small ubiquitin-like modifier protein) to the beginning of GrB. In healthy cells, this SUMO-GrB fusion remains harmless because:
The SUMO tag blocks GrB's active site, preventing it from functioning
The natural GrB activator (cathepsin C) cannot remove the SUMO tag
Only SENP1, which specifically recognizes and cleaves SUMO proteins, can liberate the active GrB1
SUMO1 (97aa) + Granzyme B = Conditional Activation
The team systematically verified their design through a series of careful experiments:
| Experimental Phase | Key Observation | Significance |
|---|---|---|
| Protein Cleavage | SENP1 converted 43 kDa SUMO-GrB to 32 kDa active GrB | Proof of concept: cancer protease can activate engineered molecule |
| Cell Death Assay | Selective apoptosis only in SENP1-overexpressing cells | Demonstrated target-specific activity with minimal off-target effects |
| Dose Response | Cell death correlated with SENP1 concentration | Established precision targeting based on protease abundance |
| Platform Validation | Successful creation of EK-responsive and TEVp-responsive GrB | Confirmed modular design adaptable to multiple protease targets |
Cell death was observed only in cells expressing the target protease (SENP1), demonstrating the specificity of the COVERT platform.
The COVERT platform successfully created molecules responsive to different proteases, confirming its adaptable design.
The COVERT platform's development required sophisticated molecular tools and techniques. Here are the key components that made this research possible:
| Research Tool | Function in COVERT Research |
|---|---|
| SUMO1 Peptide | Serves as the inhibitory domain cleavable by SENP1 protease |
| Granzyme B | Core cytotoxic component that triggers apoptosis upon activation |
| SENP1 Protease | Cancer-associated enzyme that serves as the activation trigger |
| HEK293T Cell Line | Model system for protein production and functional testing |
| Western Blotting | Analytical method to detect protein cleavage and activation |
| Primary Human T-cells | Validation system demonstrating compatibility with immunotherapy |
| Enterokinase (EK) | Alternative protease used to demonstrate platform modularity |
| Tobacco Etch Virus Protease (TEVp) | Additional protease proving design flexibility |
Western blotting, flow cytometry, and cell viability assays were crucial for validating the COVERT platform's functionality.
Recombinant DNA technology enabled the creation of fusion proteins with specific protease recognition sites.
Various cancer cell lines with different protease expression profiles helped validate the platform's specificity.
While the COVERT platform represents a revolutionary approach to cancer therapy, it's part of a broader rediscovery of granzyme biology. Once viewed solely as executors of cell death, granzymes are now recognized as having diverse functions in inflammation, tissue remodeling, and chronic diseases.
Researchers have discovered that GrB and other granzymes contribute to various conditions when dysregulated:
GrB damages corneal and conjunctival tissues in inflammatory eye conditions6
Elevated GrB contributes to impaired wound healing in aged skin and chronic inflammatory conditions
GrB promotes both choroidal neovascularization and subretinal fibrosis
Just as we can engineer GrB to target cancer cells, we might design inhibitors to block its damaging effects in chronic inflammatory conditions.
| Technology | Principle | Application |
|---|---|---|
| COVERT Platform | Engineered GrB activated by cancer proteases | Targeted cancer therapy with reduced off-target effects |
| NIR-II Ratiometric Nanoprobe | Fluorescence activation upon GrB cleavage | Non-invasive monitoring of immunotherapy response4 |
| Ga68-PET Imaging | Radiolabeled tracers binding to GrB | Tracking T-cell activation in tumors7 |
| PI-9/SERPINB9 | Endogenous inhibitor of GrB | Protecting healthy cells from immune-mediated damage3 |
The COVERT platform represents a paradigm shift in targeted therapy. By moving beyond surface-level recognition to intracellular verification, it offers a promising strategy to overcome the fundamental limitation of current treatments: their inability to reliably distinguish healthy from diseased cells.
The implications extend far beyond the initial SENP1-targeting molecule. The platform's modular nature means researchers can now design conditional GrB molecules activated by various cancer-specific intracellular signals, potentially creating tailored therapies for different cancer types.
Perhaps most excitingly, the research team has already taken preliminary steps toward integrating COVERT molecules into adoptive T-cell therapies, demonstrating that engineered human T-cells can produce, package, and deliver these smart therapeutics in response to antigen stimulation1 .
The COVERT platform is currently in the proof-of-concept stage with promising preclinical studies underway.
As we stand at this frontier of medical science, the vision of true "magic bullets" seems increasingly attainable. Through creative bioengineering and a deepening understanding of cellular biology, we're developing weapons that don't just blast everything in their path but can distinguish friend from foe at the most fundamental level—a critical advancement in our enduring campaign against cancer.