Breast cancer is a growing concern, no pun intended. But it has recently surpassed lung cancer as the most commonly diagnosed cancer in the world, and is the fifth leading cause of global cancer death. In the United States, one in eight women will develop some form of breast cancer throughout her lifetime. While every two minutes a woman is diagnosed with breast cancer, and every 13 minutes someone dies from it. That means by the end of this video in just the US, around seven to eight women will be diagnosed and one woman will die from breast cancer. And that says nothing about the friends, lovers, sisters, daughters, mothers or wives around the world who are affected. So what new developments are being investigated in treating this disease?
Hi, folks. My name is Cole and I have a Master’s of Immunology. Today on Investigate, Explore Discover, we’re going to be looking at an antibody based breast cancer therapy. So hang around with me throughout this whole video to get all of the relevant background information. So that way we can dive into some exciting experimental results.
Now in regards to breast cancer, there are many different reasons why this may happen. Some of these reasons may be genetic, where 15% to 20% of breast cancer diagnoses are familial. Additionally, 5% to 10% of breast cancer is just hereditary, whereby individual mutations like in BRCA1 or BRCA2 are significant contributors. The remaining 70% to 80% of breast cancer arises sporadically. And there are numerous risk factors that women face throughout their lives that influence this. They can include smoking, radiation exposure and excessive alcohol consumption, to name a few.
Breast cancer, like all cancers, does not strike everyone the same. To start off, there are multiple areas in the breast where cancer can occur, it can develop in the ducts, the lobes, or combination of both and be inflammatory or mucinous in nature. Now, besides where the cancer occurs, there are also common cell receptor expression patterns. The most commonly expressed receptors are progesterone or estrogen receptors, while the Her2 positive and triple negative expression patterns are more rare. Cells that express estrogen receptor usually are not as malignant and result in favorable treatment prognosis. While cells that overexpress Her2 are more difficult to treat. Triple negative breast cancer is the most difficult and deadly form of breast cancer to treat, in part because it looks just like normal cells.
Now as it stands, there are multiple different ways that we currently treat breast cancer. They involve local treatments like surgery and radiation, or systemic treatments like chemotherapy, hormone therapy and targeted therapy. These systemic treatments target many different cellular structures and pathways to try and kill the cancer cells. New therapy options like identifying new antibodies or molecules to inhibit cell receptors. And new treatment types like immunotherapy are also continuously being investigated and developed. Antibody based therapies target abundant surface receptors. CCR5 is a surface receptor that is overexpressed in several malignancies, which include breast cancer. It is present in around 50% of all breast cancer cells and over 95% of triple negative cells.
To be specific, CCR5 is a G-protein coupled transmembrane chemokine receptor, which is typically only expressed on immune cells. Chemokine receptors help coordinate the movement of cells throughout the body towards their cognate chemokines. When bound by its ligands, the chemokine CCL3, CCL4, and CCL5, CCR5 becomes activated. This causes an intracellular signaling cascade that results in intracellular calcium release, which helps to mediate cell migration and cytokine release. This calcium flux can be visualized by getting cells to co-express green fluorescent protein when that calcium release happens. As a result, you can see when these cells become stimulated by a ligand and or agonist and when they return to a resting state by observing the fluorescence signal intensity. CCR5 activation also results in many different cellular responses that are involved in cell migration and DNA repair pathways. These can be especially problematic when expressed on non-immune cells, leading to cancer progression, metastasis and therapy resistance.
Now, CCR5 is something that you might have heard about in other contexts. You might have heard about it because it is used by HIV as a coreceptor to invade host cells. It has also gathered some hype due to HIV treatments that actually block CCR5. Leronlimab, a drug created by CytoDyn, is one of these treatments. In clinical trials, this medicine suppresses viral loads in 81% of patients who have HIV, and the FDA has granted it fast track designation for its biological license application. It is a weekly subcutaneous injectable medicine that allows for easy home administration. Leronlimab itself is a humanized monoclonal antibody that competitively binds to the CCR5 receptor on white blood cells at a higher strength than HIV. Thus, it effectively blocks CCR5 tropic HIV from entering into the cells. Compared to other ways of combating HIV infections, Leronlimab has been shown to have no serious adverse effects, negligible toxicity and induces no drug resistance in hundreds of patients over the course of four years and completion of phase three clinical trials.
Now, I want to take a moment and really highlight why repurposing drugs for different diseases is so important. These CCR5 positive breast cancer cells have increased metastasis and correlate with poor prognosis. So because CCR5 inhibitors have been shown to be safe and effective against HIV infection. This enables investigators to skip phase one safety trials, because they’re already done. This gives healthcare providers a leg up on identifying another possible tool to help fight breast cancer.
This brings us to the paper that we’re focusing on today. This paper is called “Leronlimab, a humanized monoclonal antibody to CCR5 blocks breast cancer cellular metastasis, and enhances cell death induced by DNA damaging chemotherapy” by Jiao et al. from the Baruch S. Blumberg Institute at the Pennsylvania Biotechnology Center in Wynwood, Pennsylvania, USA. And in this paper the authors investigated using Leronlimab as an effective treatment for human breast cancer in a mouse model.
MDA-MB-231 is a human breast cancer cell line which is known to have a subpopulation which expresses CCR5. Because CCR5 is overexpressed on many breast cancers and are very abundant in triple negative breast cancer, the authors wanted to determine how well Leronlimab could bind to triple negative-like breast cancer cells. So to do this, they ensured that the cells over-expressed human CCR5 using an expression vector system. The authors then tested different levels of Leronlimab to determine the optimal binding efficiency. Their results showed that at levels of Leronlimab at concentrations of four micrograms per milliliter and upwards, led to almost a 98% receptor occupancy. Now, to more effectively assess the effects of Leronlimab on CCR5 function, the authors stably transfected CCR5 into these breast cancer cells. Because CCR5 activation is known to induce calcium flux, the authors next investigated calcium responses of the cells to CCL5.
The authors incubated the CCR5 expressing breast cancer cells, with or without different levels of Leronlimab. They identified that blocking CCR5 with Leronlimab resulted in a loss of calcium flux in these cells. And these graphs illustrated a little clearer across multiple experiments. Before we go on, I have to point out, like I’m always a fan of looking at microscopy because I enjoy seeing exactly what the cells look like. But if you’re going to be experimenting with a drug, spell the name correctly, and be consistent with it. But I digress. The authors also compare this calcium flux activity to a known CCR5 small molecule inhibitor, Vicroviroc, which showed the same inability to activate calcium signaling via CCL5. The authors also looked at the calcium flux of cells from other CCR5 agonists. They looked at CCL3 and found that Leronlimab inhibits CCL3 activation of CCR5, and the same CCR5 inhibitory conclusions can be drawn when stimulating with CCL4. The ability of breast cancer cells to invade extracellular matrices is distinguishable from but an important step in tumor metastasis. To determine extracellular invasion, the authors performed a type of transwell migration, which shows how much CCR5 expressing cells move towards a certain chemokine. They found that by adding Leronlimab or Vicroviroc to cells, it prevented them from moving towards CCL5.
Now this is shown in this really neat cube image over here, by the difference in cell travel to the end of the cube. However, when going through the data, I had difficulty finding the exact concentrations of reagents that they used for this particular experiment, which is typically something you really want to be clear about when reporting new findings. It was fortuitous, though, that they also tested different levels of Leronlimab to confirm their findings, and at least Leronlimab was spelled correctly in this figure.
Now, in view of the findings that CCR5 inhibition by Leronlimab reduced calcium signaling and cell invasion, the authors wanted to determine the in vivo effect of Leronlimab on lung cancer metastasis. To do this, they injected breast cancer cells that they could non invasively image into the tails of immunocompromised mice, and after eight weeks, they looked at the development of lung cancer. They found that by blocking CCR5 by either giving the mice the safe human bioequivalent dose through IP injections of Leronlimab or oral administration of the small molecule inhibitor of CCR5 Maraviroc, these treatments showed a 98% reduction in the volume of lung metastases compared to control mice.
Now, because another result of CCR5 stimulation is activation of DNA repair pathways, the authors next investigated the potential for Leronlimab to sensitize breast cancer cells to DNA damaging agents. The authors treated breast cancer cells with Doxorubicin, a topoisomerase 2 inhibitor that induces DNA damage. They found that the addition of Leronlimab decreased cell proliferation and increased cell death. This result was also found with Marovirac, and neither of these agents cause significant cytotoxicity by themselves. Additionally, these results were repeated in multiple breast cancer cell lines with the same results. After identifying this, the authors subsequently performed studies to determine whether Leronlimab could reduce the volume of established breast cancer metastasis. To do this, the authors used the same setup as before, by injecting fluorescing breast cancer cells in the tails of mice. But in this experiment, they let the cancer have a seven week headstart for growing. It was at this stage that they then looked at what treatment of Leronlimab did for reducing present metastasis and survival. They found that Leronlimab caused three out of eight mice to survive the experimental timeframe of 37 weeks, and they died at a slower rate than the control mice. In light of this, the authors wanted to figure out why exactly this might be occurring. So they looked at the lung tissues. Shown here is the non-treatment control lungs. And this is what it looks like with Leronlimab. They found that Leronlimab induced a decrease of tumor size at any point during the experiment in five out of eight mice. And in these representative images, you can see a clear decrease in the ratio of tumor cells to healthy cells with Leronlimab present, indicated by the presence of white spaces in the pictures.
Now, to quickly summarize everything altogether, the authors of this paper first took breast cancer cells, and overexpressed CCR5 on them. They found that by adding just a small amount of Leronlimab to these cells, they were able to get full receptor occupancy of CCR5 expressing cells. Cancer metastasis is induced by CCR5 expressing cells moving to areas in the body where its cognate chemokines, like CCL3, 4 and 5 are. The authors show that Leronlimab inhibits the movement of these cells in both in vitro and in vivo models. They also assessed how well Leronlimab could work with known chemotherapeutics. And while testing it with a DNA damaging chemotherapeutic doxorubicin, the authors found that Leronlimab helped to cause increased cell death and lower proliferation.
Now, not only do I think that these results are exciting to investigate and learn about, they’re also significant in a broader context. This information is significant because through their studies Jiao et al. confirm that Leronlimab effectively binds to CCR5 expressing breast cancer cells, and showed that Leronlimab administration prevents breast cancer cell metastasis and increases life expectancy of mice with CCR5 positive breast cancer. They also showed that in conjunction with the chemotherapeutic Doxorubicin, Leronlimab increases cell death. These findings suggest that additional clinical studies of Leronlimab in metastatic human breast cancers are warranted. All science is basically a stepping stone for new knowledge. And these steps are driven by questions. As always, I have a few questions myself after reviewing this information.
The first thing on my mind is how will Leronlimab affect anticancer functions of human immune cells that happen normally? Because these experiments were performed in immunocompromised mice, this data cannot tell us how Leronlimab will work in the immune system to fight off cancer. Furthermore, because this treatment was just being administered via IP injection, what would happen if Leronlimab was injected at the cancer site and then allowed to circulate throughout the body? Could it maybe reduce the necessary dose? Another question that I had revolves around other CCR5 related treatments. Other small molecule CCR5 inhibitors are effective against multiple cancer types. So would this activity also hold true for Leronlimab? I mean, we can only tell by performing more experiments. As always, my final question revolves around you. What sort of ideas or questions popped into your head when hearing about this information? I would love to hear about them in the comment section below. Also, let me know if there are any topics that you’d like to hear about in the future.
Ultimately, I hope that you learned something. But more importantly, I hope you enjoyed your time doing so. So if you did, give this video a like and subscribe for more in the future. Well, that’s everything for today. Thank you for watching, and I’ll see you next time.
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