Introduction to Probody Drug Conjugates
Antibody-drug conjugates (ADCs) have become a powerful tool in targeted cancer therapy, combining the specificity of monoclonal antibodies with the potency of cytotoxic drugs. However, traditional ADCs often target antigens such as EGFR or TROP2, which are not only present on tumor cells but also expressed to varying degrees in normal tissues. This lack of tumor specificity can lead to “on-target, off-tumor” toxicity. These side effects, which result from ADCs binding to healthy cells, may sometimes force dose reductions or even lead to treatment discontinuation.
To overcome this limitation, researchers have developed a new class of ADCs featuring conditionally active antibodies, known as Probody drug conjugates (PDCs). The design of PDCs is inspired by small-molecule prodrugs, which remain inactive during circulation and are only activated at specific disease sites to improve selectivity and reduce systemic toxicity.
Probody drug conjugates (PDCs) are engineered IgG antibodies designed to suppress their antigen-binding activity under normal physiological conditions. This can be achieved by attaching masking peptides to the antibody’s binding site via cleavable linkers, or by engineering binding domains that undergo pH dependent conformational changes. Once the probody reaches the tumor microenvironment, which is typically rich in proteases and has a slightly acidic pH, the mask is removed or the structure shifts, reactivating the antibody’s ability to bind its target and deliver its cytotoxic payload precisely within the tumor. These two strategies represent the main routes of conditional activation in PDCs.
Figure 1. Probody drug conjugates.
Protease-Cleavable Masking Strategies
One of the most common approaches for conditional activation in PDCs involves attaching self-masking peptides to the antibody, which are designed to be cleaved by proteases commonly found in the tumor microenvironment. These proteases, such as urokinase-type plasminogen activator and membrane-type serine proteases, are usually present at low levels in healthy tissues but are significantly upregulated in many cancers.
A notable example is CX-2029. CX-2029 targets CD71, a protein highly expressed on tumor cells but also found on normal tissues, making it challenging for traditional ADCs. To address this, CX-2029 uses a self-masking peptide fused to the antibody’s binding site through a protease-cleavable linker. This masking peptide blocks the antibody’s ability to bind CD71 while circulating in the body. Once CX-2029 reaches the tumor microenvironment, which is rich in specific proteases, these enzymes cleave the linker and remove the masking peptide. This cleavage reactivates the antibody, allowing it to bind CD71 on tumor cells and deliver its cytotoxic payload-MMAE, directly to the cancer.
Preclinical studies showed that CX-2029 has strong anti-tumor activity comparable to traditional ADCs but with a much higher maximum tolerated dose in animal models, indicating a wider therapeutic window and reduced toxicity. Early clinical trials involving patients with various advanced solid tumors have demonstrated promising anti-cancer activity with manageable side effects.
The development of CX-2029 highlights how protease -activated masking peptides can effectively improve tumor selectivity and safety, opening the door to targeting antigens that were previously considered too risky due to their presence on healthy tissues.
Figure 2. Protease-Cleavable Masking Strategies a. Illustrative structure of CX-2029. b. Schematic illustration of CX-2029 in non-malignant cell. c. Schematic illustration of CX-2029 activation in tumor cells.
pH-Triggered Activation Strategies
The tumor microenvironment is typically more acidic (pH 6.0 - 6.8) than healthy tissues (pH 7.3 – 7.4), and this difference can be used to activate certain PDCs through pH-dependent structural changes in the antibody. A common approach is to introduce histidine residues into the binding regions, allowing the antibody to bind its target only under acidic conditions.
One representative example of a pH-sensitive PDC is HTI-1511 targeting EGFR. The antibody’s binding site is engineered to include histidine residues, which have a unique pKa around 6.0. Under the mildly acidic conditions of the tumor microenvironment, these histidines become protonated, leading to a conformational change that increases the antibody’s affinity for EGFR. Conversely, at normal physiological pH, the histidines remain mostly unprotonated, resulting in weaker antigen binding. This pH-dependent binding mechanism enables HTI-1511 to selectively target EGFR-expressing tumor cells while minimizing interaction with healthy tissues, thereby improving its therapeutic window.
In preclinical models, HTI-1511 showed robust activity against EGFR-expressing tumors, including those resistant to cetuximab or with KRAS/BRAF mutations, with minimal binding to normal tissues. It was well tolerated in non-human primates. Although no active clinical development has been reported since 2018, HTI-1511 illustrates how pH-tuned binding can enhance tumor specificity and reduce toxicity in ADC design.
Figure 3. pH-Triggered Activation Strategies a. Illustrative structure of HTI-1511. b. Schematic illustration of HTI-1511 in non-malignant cell. c. Schematic illustration of HTI-1511 activation in tumor cells.
Conclusion
Probody drug conjugates represent a promising evolution in ADC technology, offering greater tumor specificity through environment – sensitive activation. By masking antibody activity until the drug reaches the tumor microenvironment, PDCs aim to minimize off-target toxicity and expand the range of targetable antigens. While clinical development is still ongoing, early examples like CX-2029 and HTI-1511 highlight the potential of this strategy to improve both safety and efficacy in cancer treatment. Continued research and innovation in this field may pave the way for a new generation of smarter, more selective cancer therapies.
Reference
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