tumor cells Archives | Mikhail Blagosklonny Oncotarget

“This study focuses on developing a new generation of antibodies that can get inside cancer cells and disrupt their DNA repair processes, offering a hopeful new way to treat cancer with more precision.”

Cancer research has made remarkable progress in recent years, with monoclonal antibody (mAb) therapy emerging as one of the most promising advancements. These treatments are designed to precisely target cancer cells, offering a more focused approach that helps patients fight different malignancies with fewer side effects compared to traditional chemotherapy.

Despite this progress, a major challenge remains: targeting cancer-related molecules inside cells rather than on the surface, which has been the main focus of available mAb therapies until now. This is where the groundbreaking research in the paper “Next-generation cell-penetrating antibodies for tumor targeting and RAD51 inhibition,” published in Volume 15 of Oncotarget on October 1, 2024, comes into play.

The Study

This study, led by researchers Madison Rackear, Elias Quijano, Zaira Ianniello, Daniel A. Colón-Ríos, Adam Krysztofiak, Rashed Abdullah, Yanfeng Liu, Faye A. Rogers, Dale L. Ludwig, Rohini Dwivedi, Franziska Bleichert, and Peter M. Glazer from Yale University School of Medicine, Yale University, and Gennao Bio, explores the potential of an innovative mAb called 3E10. This antibody offers a new way to target cancer cells. The researchers focused on creating humanized versions of 3E10 that can enter malignant cells and disrupt their DNA repair system, presenting a promising new approach to cancer treatment. In this blog, we will look at the key findings and implications of this important work.

The Challenge: Targeting Intracellular Molecules

To understand the importance of this research, let’s first look closer at the limitations of conventional monoclonal antibodies. mAbs are proteins designed to bind to specific targets, like a key fitting into a lock. Many of the current mAb therapies work by targeting proteins (antigens) found on the surface of cancer cells. The issue is that not all cancer-related targets are located on the cell surface. In fact, many important proteins that drive malignant tumor growth and therapy resistance are found inside the cells. Until now, it has been difficult to develop therapies that can penetrate the cell membrane and reach these intracellular targets. The few antibodies that can do this usually face degradation inside the cell, meaning they lose their power before they reach their intended target.

3E10: A Unique Antibody with Cell-Penetrating Abilities

The researchers in this study focused on 3E10, a monoclonal antibody (mAb) originally discovered in a mouse model used to study systemic lupus erythematosus, an autoimmune disease. Unlike most antibodies, 3E10 can enter cells and even reach the cell’s nucleus. What makes it unique is that it does not rely on the usual pathway most antibodies use to enter cells, which usually leads to them being inactivated in cellular compartments called lysosomes. Instead, 3E10 enters cells through a nucleoside transporter called ENT2, which is highly active in many cancers. This overactivity happens because cancer cells grow and multiply quickly, requiring extra nucleosides, the building blocks of DNA and RNA.

The way 3E10 enters cells makes it an exciting candidate for cancer therapy because it can reach targets inside cancer cells that are typically hard to access. One key target is a protein called RAD51, which is crucial for repairing damaged DNA. By binding to and blocking RAD51, 3E10 prevents cancer cells from repairing their DNA, making them more vulnerable.

Humanizing 3E10: Creating Antibodies Suitable for Human Use

While 3E10 holds great potential, the original version of the antibody was derived from mice, which is a problem for human therapy. Antibodies from other species can activate an immune response in humans, leading to reduced efficacy and side effects. To overcome this, the researchers aimed to “humanize” the antibody. This process involved modifying the 3E10 so that it closely resembles a human antibody, minimizing the risk of immune rejection.

In this study, researchers developed 22 different humanized versions of 3E10, each with modifications designed to increase its ability to enter cells and bind to nucleic acids (such as DNA and RNA). These variants were then tested to evaluate how effectively they could do so.

The Results

Tuning Antibody Properties for Optimal Cancer Targeting

The researchers discovered that different humanized versions of 3E10 showed different abilities to bind nucleic acids and penetrate cells. One variant, called V66, stood out for its high affinity for nucleic acids and strong ability to enter cells. In contrast, another variant, V31, had lower affinity for nucleic acids but showed higher binding to RAD51 (a DNA repair protein) and was also more effective at inhibiting DNA repair mechanisms in cancer cells that already had DNA repair problems.

These findings suggest that by adjusting the characteristics of 3E10, it is possible to create different versions of the antibody for different treatments. For instance, the V66 variant may be more suitable for delivering therapeutic molecules into cells because it enters them more efficiently, while lower-affinity variants like V31 might be better at directly blocking the DNA repair mechanisms in cancer cells.

Tumor Targeting Without Antigen Dependence

One of the most promising aspects of this research is that the 3E10 antibody can target malignant tumors without needing a specific protein on the surface of cancer cells. Instead, 3E10 detects tumors because of the high levels of certain molecules, like nucleosides and DNA, commonly found in cancer tissues. This gives a big advantage over many current treatments, which focus on specific proteins found on cancer cells. These proteins can vary in how much they are present between patients or even between different parts of the same tumor, making those treatments less reliable.

Therapeutic Potential

The ability of 3E10 to enter cells and block a crucial DNA repair protein like RAD51 makes it a strong candidate for treating different cancers, including breast, ovarian, and prostate cancers. Additionally, 3E10 can be modified for other purposes, opening up many possibilities for future cancer treatments. For instance, the study’s authors suggest that humanized 3E10 could also be used as a tool for delivering genetic material into cells for gene therapies. This could help create more personalized and effective cancer treatments in the future.

Conclusion

This study represents an important step in developing a new generation of mAb for cancer treatment. By humanizing and optimizing the 3E10 antibody, researchers have shown its potential to target cancer cells in new ways from the previously used. Whether it is used to prevent cancer cells from repairing their DNA or to deliver drugs directly into tumors, 3E10 is a promising new tool in the fight against cancer.

As cancer therapies continue to improve, innovations like 3E10 offer hope for more precise and effective ways to target even the toughest cancers. However, further testing will be needed to make sure these new-generation antibodies are safe and effective in humans.

Click here to read the full research paper in Oncotarget.

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In this study, researchers show that treatment of 4T1-12B mouse breast cancer cells with this nanobody inhibits V-ATPase-dependent acidification of the media and invasion of these cells in vitro.

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Researchers recently developed a nanobody directed against an extracellular epitope of the mouse V-ATPase c subunit. Zhen Li, Mohammed A. Alshagawi, Rebecca A. Oot, Mariam K. Alamoudi, Kevin Su, Wenhui Li, Michael P. Collins, Stephan Wilkens, and Michael Forgac from Tufts University School of Medicine; Tufts University; Dana Farber Cancer Institute, Harvard Medical School; University of Minnesota School of Medicine; Prince Sattam Bin Abdulaziz University; Korro Bio; SUNY Upstate Medical University; and Foghorn Therapeutics, suggest that plasma membrane V-ATPases represent a novel therapeutic target to limit breast cancer metastasis. The vacuolar H+-ATPase (V-ATPase) is an ATP-dependent proton pump that functions to control the pH of intracellular compartments as well as to transport protons across the plasma membrane of various cell types, including cancer cells.

On August 14, 2024, their research paper was published in Oncotarget’s Volume 15, entitled, “A nanobody against the V-ATPase c subunit inhibits metastasis of 4T1-12B breast tumor cells to lung in mice.”

The Research

Breast cancer is one of the most diagnosed cancers, accounting for almost one-third (30%) of all new diagnoses in women in 2022. At the time of diagnosis, 20–30% of patients with early-stage breast cancer will go on to develop metastatic breast cancer. 6–10% of all patients with breast cancer have stage IV disease at time of diagnosis. It has been shown that V-ATPase plays an important role in promoting the invasiveness of many cancer cell types, including breast cancer cells.

This study demonstrated that inhibiting cell surface V-ATPases can effectively block tumor cell invasion. The findings indicate that anti-V-ATPase antibodies targeting an extracellular region of the V-ATPase can suppress activity on the surface of cancer cells, as well as inhibit both in vitro invasion and in vivo metastasis in a mouse model. This represents a promising advancement toward developing a new therapy to limit breast cancer metastasis.

Results

A camelid nanobody against the N-terminus of the mouse V-ATPase c subunit was prepared using phage display. The nanobody was dimerized through disulfide bonding to create a bivalent molecule. The purified nanobody was detected using Coomassie blue staining and Western blotting. The apparent molecular weight of the dimer on SDS-PAGE was around 45 kDa, slightly faster than the predicted weight of 56.8 kDa. The nanobody was tested for its ability to inhibit V-ATPase-dependent acidification in mouse 4T1-12B cells. The nanobody treatment resulted in a similar increase in extracellular pH as treatment with concanamycin, a known V-ATPase inhibitor.

Combining both the nanobody and concanamycin did not significantly enhance the effect. The nanobody effectively inhibited V-ATPase-dependent extracellular acidification without affecting cell viability. The anti-V-ATPase nanobody was tested for its ability to inhibit in vitro invasion of 4T1-12B cells. Treatment with the nanobody significantly inhibited invasion, like its inhibition of extracellular acidification. The nanobody effectively inhibits both extracellular acidification and in vitro invasion of 4T1-12B cells with similar affinity.

The administration of the anti-V-ATPase nanobody was tested to determine its effect on tumor growth and metastasis in mice. Different amounts of the nanobody were administered to mice without any adverse effects. The effect of nanobody administration on in vivo metastasis was then tested using 4T1-12B cells implanted in the mammary fat pad. However, no significant difference in tumor volumes was observed between the control and nanobody-treated groups at the end of the study. Treatment with the anti-V-ATPase nanobody resulted in a significant reduction in lung metastasis but had no effect on tumor growth or leg metastases. No significant metastasis was observed in other organs. In contrast, treatment with the anti-GFP nanobody did not reduce lung metastases.

Discussion

The researchers’ previous results demonstrated that selective inhibition of cell surface V-ATPases using an antibody or bafilomycin showed potential in inhibiting invasion of breast cancer cells. However, the use of antibodies against the native c subunit proved challenging due to its conservation and limited exposure. To overcome this, a nanobody against a native epitope of the c subunit was developed through in vitro screening. This nanobody successfully inhibited cell surface V-ATPase activity in mouse 4T1-12B breast cancer cells and showed a correlation between inhibition of invasion and extracellular acidification. In mice, the nanobody treatment significantly reduced lung metastases, but had no effect on tumor growth or leg metastasis.

The study suggests that different mechanisms may be involved in tumor cell invasion in different tissues. The potential side effects of inhibiting cell surface V-ATPases were also discussed, highlighting the limited presence of these pumps in certain cells and the potential benefits of inhibiting osteoclast function for breast cancer metastasis to bone.

Overall, the findings support the use of inhibitory nanobodies against cell surface V-ATPases as a potential therapeutic approach to inhibit breast cancer metastasis.

“These results provide support for the use of an inhibitory antibody directed against an extracellular epitope of the V-ATPase as a potential anti-metastatic therapeutic to inhibit breast cancer metastasis.”