Targeting EGFR to enhance NKT cell-mediated killing of lung cancer cells

 

 

What you will learn in this on-demand immuno-oncology webinar:

 

>> Current status of non-small cell lung cancer treatment

>> Cell therapy using NKT cells and in combination with EGFR inhibitors

>> Cancer spheroid models and the importance of modeling the tumor microenvironment

>> Using the Maestro Z live-cell analysis platform to evaluate potency of cell therapies against cancer spheroids and monolayers, label-free and in real time

 

About the presenters:

 

 

 

Transcript of webinar:

 

Abigail Pinchbeck: Hello and a very warm welcome to today's Cell and Gene Therapy Insights webinar titled "Targeting EDFR to Enhance NKT Cell-Mediated Killing of Lung Cancer Cells." I'm Abby Pinchbeck, an editor at BioInsights, and joining me today are Tonya J. Webb, David Ferrick, and Stacie Chvatal. They will delve deeper into their case study testing the hypothesis that EGFR treatment reduces cancer-mediated immunosuppression, sensitizing the tumor cells to NKT-mediated cytolysis.

After the presentation, we'll have a live Q&A session with Tonya and Stacie, where we invite our audience to post their questions to our speakers using the "Ask a Question" box at the bottom of your screen, and we'll try to get to these during the session. I'd also like to draw your attention to the Resources tab on the right, where you can find more information on the topics covered today. Now, I'd like to introduce our presenters.

Dr. Tonya J. Webb is a tenured associate professor at the Department of Microbiology and Immunology at the University of Maryland School of Medicine. She completed a doctoral degree in Microbial Immunology at Indiana University, with studies focused on investigating the role of CD1d1 molecules and NKT cells in antiviral immunity.

Stacie Chvatal is the Director of Product Management at Axion BioSystems. She earned a Bachelor of Science in Engineering from Mercy University and a PhD in Biomedical Engineering from the Georgia Institute of Technology and Emory University.

Dr. Tonya J. Webb: Hi, my name is Tonya Webb, and I'm so happy to have the opportunity to share with you some of the work from my lab focused on targeting epidermal growth factor receptor to enhance NKT cell-mediated killing of lung cancer. These are my disclosures.

Our lab focuses on a unique population of T cells called natural killer T cells or NKT cells. These cells display characteristics of both the innate and adaptive arms of the immune system. So, like innate cells, once activated, NKT cells can rapidly exert their effector functions, and they display proteins that are characteristic of natural killer cells. However, like T cells, NKT cells are activated through their T Cell receptor. In contrast to classic T cells, which recognize antigens presented in the context of MHC molecules, NKT cells are activated by lipid antigens presented in the context of CD1d. A classic antigen used is Alpha-galactosylceramide or Alpha-GalCer.

What's important is that NKT cells have been evolutionarily conserved. So NKT cells are very similar across individuals. Therefore, if we develop a therapy targeting NKT cells, it can be used across many different populations due to their conserved nature. NKT cells are a promising target for cancer immunotherapy. This is because NKT cells can promote anti-tumor immunity in a variety of ways. Once activated, NKT cells can directly mediate cytotoxicity or kill cancerous cells. Not only that, following their activation, they rapidly secrete cytokines that can help activate natural killer cells, as well as induce the maturation of dendritic cells. These dendritic cells can then license cytotoxic T lymphocytes.

While they do hold great promise, their efficacy has been limited because NKT cells are suppressed in many cancer patients, and so incorporation into clinical trials has been limited. We know that NKT cells can play a key role in anti-tumor immunity based on studies like this from Dale Godfrey's group, in which they compared tumor growth in wild-type and NKT cell-deficient animals. If you look here and compare wild-type mice shown in the black squares to the open squares in mice which lack NKT cells, you see rapid tumor growth in mice lacking NKT cells. In contrast, if you give NKT cells back, you see a dose-dependent reduction in tumor growth.

I would like to reiterate that NKT cells are a promising target for cancer immunotherapy if we understand how to harness their potential. Because while NKT cells can directly kill tumor cells and activate other immune cells, NKT cells are reduced in cancer patients. So, this is data from our Cancer Center where we compared healthy donors, patients with lymphoma, breast cancer, and prostate cancer. What I think you can appreciate is that across the board, NKT cells are reduced in number in patients. And these are just looking at NKT cells in the blood.

However, studies from other groups have shown that if you treat with NKT cell-based therapy, this can result in a reduction in tumor progression in multiple myeloma patients, and this occurred in three out of four patients. In addition, in patients with head and neck cancer, all 10 patients that completed the trial had stable disease or either a partial response five weeks after the initiation of NKT cell-based therapy. Not only that, in lung cancers, it was found that 60 percent of advanced lung cancer patients with highly active NKT cells had an increased median survival time. The median survival time was 29 months compared to patients that received the standard of care, in which their median survival time was only 4.6 months. So, again, if we learn how to effectively modulate or activate NKT cells, they are really a key target for cancer immunotherapy.

Now, you may be wondering, how can we restore or enhance NKT cell responses in cancer patients? And you may also be wondering, how does the cancer grow when you have an immune system that can recognize and destroy cancerous cells? This leads me to the theory of cancer immunoediting, which has three phases: elimination, equilibrium, and escape.

Initially, when you have malignant cells, these cancerous cells can be killed by your immune system. However, sometimes these cancer cells are allowed to persist, and a state of equilibrium is reached wherein the cancer cell will down-regulate antigens and molecules that their immune system uses to recognize these cancerous cells. Then, the cancer cells will continue to undergo genetic alterations, express inhibitory ligands, secrete suppressive factors, as well as recruit immunosuppressive cells. This is when they are allowed to escape immune pressures. So, when a patient comes to the clinic, it's in this escape phase wherein the immune system is no longer able to control the growth of the cancer.

So, this is what we're focused on once the cancer has escaped immune pressures. We're wondering how can we target these cancers? Can we enhance NKT cell responses to lung cancer once the cancer has escaped these pressures in these studies?

We are focused on lung cancer because lung cancer is the most lethal and second most diagnosed type of cancer in American men and women. In addition, non-small cell lung cancer accounts for 85 percent of lung cancer cases, and 40 percent of non-small cell lung cancer patients have adenocarcinoma. So, we use the A549 adenocarcinoma line in our pilot studies, and we wanted to compare traditional treatments to newer approaches.

In addition to classic treatments like surgery, chemotherapy, and radiation, there are newer treatments like immunotherapy that use the patient's immune system to fight the cancer, such as our studies using NKT cells. And there are other treatments that can specifically target a patient's tumor based on changes unique to that tumor. For example, many lung cancers use epidermal growth factor (EGFR) to grow. When epidermal growth factor binds to its receptor, the receptor homodimerizes or heterodimerizes with other family members, leading to tumor cell proliferation, apoptosis evasion, angiogenesis, and metastasis.

As a result, epidermal growth factor has been a major target for treating advanced non-small cell lung cancer. For example, gefitinib and erlotinib in combination with cisplatin are first-line treatments for wild-type and mutated EGFR non-small cell lung cancer. While EGFR tyrosine kinase inhibitors such as gefitinib and erlotinib are initially effective, almost all non-small cell lung cancer patients treated with EGFR tyrosine kinase inhibitors eventually develop acquired resistance. EGFR T790M is the most common mechanism of acquired resistance, followed by mesenchymal epithelial transition protein amplification, HER2 amplification, and small cell histologic transformation.

Because patients develop resistant mechanisms like the T790M mutation, our goal was to test the effectiveness of chemotherapeutic agents like cisplatin in combination with EGFR inhibitors, a targeted therapy, along with immunotherapy.

Our overarching hypothesis was that inhibition of epidermal growth factor signaling, in combination with immune checkpoint inhibitor therapy, would reduce tumor-mediated immune suppression and lead to enhanced NKT cell killing of the tumor cells. So, in these pilot studies, we first wanted to determine if there was synergy between DNA damaging agents and EGFR tyrosine kinase inhibitors in the treatment of lung cancer.

In these studies, we used the A549 cell line and assessed cell viability using the MTT assay. If you compare the graph on the left, wherein cells were treated with cisplatin alone, you can see there was a dose-dependent reduction in viability. However, if you compare treatment with cisplatin and gefitinib alone to the combination treatment, you will see that the combination treatment induces greater cytotoxicity.

So, we first asked if EGFR tyrosine kinase inhibitors work synergistically with DNA damaging agents to enhance killing of lung cancer cells. To do this, we performed dose-response curves with TKIs to determine if viability decreases when used in combination with cisplatin. Similarly, we found that combination treatment with cisplatin and erlotinib resulted in greater cytotoxicity than treatment with either drug alone.

It's known that EGFR and Vascular Endothelial Growth Factor (VEGF) receptor can share similar downstream signaling pathways. In addition to its role in promoting angiogenesis, VEGF can also potentiate an immunosuppressive tumor microenvironment. So, we were wondering if when we treat with the EGFR tyrosine kinase inhibitors, we are also modulating VEGF.

For example, we have examined the impact of VEGF on T cell and NKT cell activation. On the left panel, I'm showing you DN32.D3 cells, which is a mouse NKT cell hybridoma that was pre-treated with VEGF and then cultured with antigen-presenting cells. What I think you can appreciate is that we saw a dose-dependent reduction in IL-2 production by NKT cells when they were treated with VEGF. Not only that, when we looked at primary cells by isolating liver mononuclear cells from C57 black 6 mice and stimulating T cells with either anti-CD3/CD28 beads or NKT cells with CD1d alpha-galactosylceramide-loaded beads.

We found a significant reduction in their production of interferon gamma. Not only that, when we used human PBMCs, we found a similar decrease in response following treatment with VEGF. These data demonstrate that VEGF can directly inhibit both NKT cell and total T cell activation.

Now, we go back to the question: Does treatment with EGFR inhibitors reduce VEGF production by A549 cells? Interestingly, we found that treatment with all three drugs examined - cisplatin, gefitinib, and erlotinib - resulted in a decrease in VEGF production by A549 cells. This happened whether they were grown in a 2D monolayer or in 3D spheroids.

We have observed synergy between DNA damaging agents and EGFR TKIs in the treatment of A549 cells. Not only that, we found a reduction in VEGF production. So, now we wanted to compare the efficacy of combination therapy in 2D and 3D models. We wanted to study the impact of our treatment strategy using a more physiologically relevant model, and 3D spheroids have higher therapeutic resistance than 2D monolayers. This is because the tumor microenvironment can be more hypoxic in a 3D model system.

In the next set of studies, we asked if the combination therapy was effective in 3D culture models. To create our 3D models, A549 cells were plated in ultra-low attachment plates for four days. We added the treatments for 72 hours and then added propidium iodide and Hoechst stain for fluorescence.

These are representative images showing that following drug treatment, the majority of the cells are dead. In these studies, A549 spheroids were treated with cisplatin, gefitinib, or in combination, and then stained with PI and Hoechst. What we found was that treatment with cisplatin and gefitinib resulted in greater cell death compared to treatment with gefitinib alone.

These studies made us realize that we need more sensitive methods to reliably detect viability. We initially assessed viability using MTT and PI/Hoechst staining; however, the quantitative assessment was modest at best. So, we tested the sensitivity levels of additional assays, the CellTiter-Glo assay as well as the Maestro impedance assay to really highlight why we needed more sensitive methods like the Maestro impedance assay.

Please look closely at these data that you've seen before. While in the images above, you can clearly see that most of the cells are dead, this is not reflected in the viability curve highlighted by the arrow. Using the CellTiter-Glo assay to assess viability in our A549 spheroids demonstrated that combination treatment resulted in greater levels of cytotoxicity. Importantly, we also found that CellTiter-Glo was much more sensitive than PI/Hoechst.

In addition, when we compared our 2D cultures to our 3D models, we found that the 3D spheroids were much more resistant to cisplatin than a 2D monolayer. Similarly, we found that the 3D spheroids were much more resistant to cisplatin and erlotinib than a 2D monolayer. This was true for both erlotinib and gefitinib.

If you look here and again compare our 2D cultures to the 3D cultures, the 3D spheroids were much more resistant to treatment.

Now that we've developed our model systems, we're back to our original question: Can we enhance NKT cell responses to lung cancer? It has previously been shown that combination treatment with EGFR tyrosine kinase inhibitors and immune checkpoint blockade can increase immune cell proliferation. In these studies, proliferation was measured by assessing Ki-67 expression, and the authors examined CD8 T cells, CD4 T cells, NKT cells, and Gamma Delta T cells.

What I really want you to focus on are these bars in yellow and green. What you can appreciate is that treatment with the EGFR tyrosine kinase inhibitor erlotinib, in combination with anti-PD-1, resulted in a significant increase in Ki-67 expression. Not only that, there's currently a phase one-phase two clinical trial ongoing to investigate the efficacy and safety of NKT cells in combination with gefitinib for the treatment of advanced EGFR-mutated non-small cell lung cancer. This group reported data on one patient. 

So, in this study, a patient diagnosed with stage four adenocarcinoma was treated with chemotherapy and bevacizumab, an antibody against VEGF. Then the patient received afatinib treatment and then gefitinib in combination with NKT cell therapy. After that, the patient received resection of the right lower lobe of the lung and had a stage one diagnosis. Later on, the patient had to stop receiving NKT cell-based therapy due to issues with the pandemic. However, they were able to continue treatment later, and importantly, the patient is still alive. Our in vitro assays will allow us to examine the mechanisms responsible for this.

In these studies, we moved forward with the Maestro impedance platform. We plated A549 cells in low attachment plates, and spheroids grew. Then we transferred the spheroids to the Maestro plates, where the spheroids will attach and resistance and viability will increase. Next, we can add either drugs or NKT cells or both, which would result in cytotoxicity, and then we can measure this using the impedance assay.

Using the Maestro system, we are able to measure viability by measuring resistance. So, we conducted a study to look at specificity. In these studies, the A549 spheroids were treated with EGFR inhibitors plus or minus epidermal growth factor (EGF). We found that spheroid viability was inhibited when we treated with the EGFR tyrosine kinase inhibitors, regardless of the presence or absence of EGF.

Now that we have a system in place, we can finally investigate the effectiveness of combination therapy, chemotherapy, targeted therapy, and immunotherapy in our 3D models. First, a little background on immune checkpoint inhibitors: the immune system is regulated by a system of checks and balances. The immune system wants to stay in homeostasis, so once you have a strong response, there are signals to dampen that response. For example, naive and memory T cells express high levels of the cell surface protein CD28, which provides a positive co-stimulatory signal. But they do not express CTLA-4.

Following activation through the T cell receptor, CTLA-4 is quickly upregulated. The stronger the stimulation through the T cell receptor and CD28, the higher the amount of CTLA-4 that is upregulated onto the T cell surface. This happens in the periphery. In contrast, within the tissues, the major role of the programmed cell death protein PD-1 or the PD-1 pathway is to regulate inflammatory responses. Activated tissues will upregulate PD-1, and they will continue to express it in the tissues. Inflammatory signals in the tissues will induce the expression of PD-1 ligands, which downregulate T cell activation. So, immune checkpoint inhibitors are antibodies that target these negative signals in order to restore T cell responses to tumors.

There are several immune checkpoint inhibitors in development. The inhibitory and stimulatory proteins, which are currently being targeted by FDA-approved monoclonal antibodies or next-generation immunotherapeutic drugs, are highlighted by red and green dots, respectively.

So, remember our original question was to see if we could restore NKT cell responses or enhance their responses to lung cancer. We wanted to check for these positive and negative signals, being that the negative signal could be blocked.

NKT cells are activated by lipids presented in the context of CD1d, so we first checked to see if A549 cells express CD1d. As shown here in the black histogram, we observed high cell surface expression of CD1d. We also examined PD-L1 levels and found that the A549s expressed intermediate levels of PD-L1.

In this set of experiments, we treated A549 cells with drugs in the presence of increasing numbers of NKT cells expanded from a healthy donor, as shown on the x-axis. While we observed a reduction in viability following treatment, the addition of anti-PD-L1 did not appear to enhance cytotoxicity. In contrast, when A549 cells were treated with increasing concentrations of gefitinib, the addition of NKT cells further enhanced cytotoxicity. So, if you'll focus here and compare the green line, which includes NKT cells, to the black line, you see that just the addition of 20,000 NKT cells resulted in an increase in cell death.

Following activation through the T cell receptor, CTLA-4 is quickly upregulated. The stronger the stimulation through the T cell receptor and CD28, the higher the amount of CTLA-4 that is upregulated onto the T cell surface. This happens in the periphery. In contrast, within the tissues, the major role of the programmed cell death protein PD-1 or the PD-1 pathway is to regulate inflammatory responses. Activated tissues will upregulate PD-1, and they will continue to express it in the tissues. Inflammatory signals in the tissues will induce the expression of PD-1 ligands, which downregulate T cell activation. So, immune checkpoint inhibitors are antibodies that target these negative signals in order to restore T cell responses to tumors.

There are several immune checkpoint inhibitors in development. The inhibitory and stimulatory proteins, which are currently being targeted by FDA-approved monoclonal antibodies or next-generation immunotherapeutic drugs, are highlighted by red and green dots, respectively.

So, remember our original question was to see if we could restore NKT cell responses or enhance their responses to lung cancer. We wanted to check for these positive and negative signals, being that the negative signal could be blocked.

NKT cells are activated by lipids presented in the context of CD1d, so we first checked to see if A549 cells express CD1d. As shown here in the black histogram, we observed high cell surface expression of CD1d. We also examined PD-L1 levels and found that the A549s expressed intermediate levels of PD-L1.

In this set of experiments, we treated A549 cells with drugs in the presence of increasing numbers of NKT cells expanded from a healthy donor, as shown on the x-axis. While we observed a reduction in viability following treatment, the addition of anti-PD-L1 did not appear to enhance cytotoxicity. In contrast, when A549 cells were treated with increasing concentrations of gefitinib, the addition of NKT cells further enhanced cytotoxicity. So, if you'll focus here and compare the green line, which includes NKT cells, to the black line, you see that just the addition of 20,000 NKT cells resulted in an increase in cell death.

I also would like to thank the Axion team for allowing us to demo the Maestro. We were able to get amazing work done within a relatively short period of time.

I would like to thank you for your time and attention and would be happy to answer any questions that you may have. Thank you so much.

Abigail: Thank you, Tonya, for that great presentation. We're now going to start our Q&A session with Tonya and Stacie, so do feel free to send in any questions that you may have for them. We will try to get to these during the session.

Tonya and Stacie, if you'd like to join me back on camera, I've got the first question here, and this one's for you, Tonya: What are some of the challenges of working with a 3D model?

While we're waiting for Tonya to respond, I'll move on to a question that we have for Stacie: "How do you know the signal that you're recording on the Maestro Z is from the cancer cells and not the immune cells?"

Stacie: Thanks, Abigail. That's a great question. The impedance measurement is actually sensitive only to the cells that are attached to the electrode in the bottom of the well. So, for example, in Dr. Webb's experiments, the lung cancer cells or the 3D spheroids were attached to the electrode, and those are what are generating the impedance signal. The immune cells, which are non-adherent, generate very little to no impedance signal when they settle down and just rest at the bottom of the well but aren't truly attached. So you can be confident that the changes in impedance that you're observing in an experiment like this are due to the behavior of the adherent target cells and not the immune cells.

Tonya: But that said, I'd like to add that actually, you can use impedance to run a similar potency assay using a non-adherent target cell or a liquid tumor model. In order to do that, you would first attach the target cells to the electrode using an antibody coating to tether those cells to the surface. Once they're tethered and in close proximity to the electrode, you will run the assay just as you can with adherent cells, and you'd see an increase in resistance as they grow and a decrease in resistance as they die off or are killed by the treatment.

Abigail: Yeah, that makes sense. Thank you so much, Tonya. Looks like, Stacie, it looks like we've got Tonya back now. So Tonya, I've got a question here for you: What are some of the challenges of working with the 3D model?

Tonya: Yes, so we really enjoyed working with the 3D model, but it could be a little challenging because it was hard to get an equal number of cells per well. So, we tried different strategies of adding multiple spheroids – we tried combinations of two or four. And also, there's a seating effect when you plate the cells. This spheroid grows out, and so you have this outer rim. And so, in being able to have a representative grouping of cells could be a little bit challenging, but we were able to work around this.

Abigail: Great, thank you. And another one for you, Tonya: "Have you considered creating more complex spheroids with additional cell types, matrices, or other aspects of the tumor microenvironment?"

Tonya: Yeah, so actually we have. The data that we focused on here was on lung cancer, but we've also done multicellular spheroids with ovarian cancer. When we use fibroblasts as a stromal support system in combination with the tumor cells, we could add in the drug and NKT cells, and we were able to get really nice data using those other models.

Abigail: Great to hear. And are you exploring NKT cell therapy for other cancers in addition to lung cancer?

Tonya: Yes, as I just alluded to, we're doing some work with ovarian cancer. We have similar studies with breast cancer, and we also work on some hematological malignancies like lymphoma, B-cell lymphoma.

Abigail: Great. And why do you think anti-PD-L1 did not have any effect?

Tonya: Also, we think that the lack of seeing differences with using anti-PD-L1 could be for numerous reasons. We're not sure if, within our spheroids, PD-L1 was downregulated when we added in the NKT cells. And if it was downregulated, then we wouldn't see that big of an effect.

Tonya: That's a great question, thanks so much. So, we really think that there are multiple inhibitory factors that the cancer is using to evade NKT cell detection. And so, we think that the reason why we're seeing this enhanced killing is not only because we're targeting EGFR, but we're also getting a decrease in VEGF. So, the tumor isn't able to proliferate or grow as well because we're targeting VEGF and EGF, and this is also decreasing the amount of immune suppression that the cancer is able to induce. And so, we think that we're able to target multiple factors using this approach, and that's why we see this enhanced killing.

Abigail: Yeah, that makes sense. And back to you here, Stacie. "All cells in a spheroid are not attached to the surface, so how accurate for the impedance of spheroids?"

Stacie: Yes, that's a great point. Of course, in a 3D model like this, only the bottom portion of that spheroid is attached to the electrode, and that's the part we're measuring from. But we've found that the T cells, or immune cells like NKT cells in this case, tend to attack that spheroid from all sides, and you see the spheroid shrink accordingly as it's getting killed by those cells. And the impedance measurement is directly correlated with the size of the spheroid. So, as the spheroids are bigger or growing, you see an increase, and as the spheroids are shrinking or being killed, you see a decrease. So, we feel confident that we are measuring accurately the killing of that spheroid even with this 2D planar electrode.

Abigail: Great. And back to you now, Tonya. Since there are several anti-VEGF therapies on the market, do you think any of these may have similar effects as the EGFR inhibitors?

Tonya: Well, we've tried several VEGF inhibitors. We've tried bevacizumab, the antibody, and we've also tried genistein, a tyrosine kinase inhibitor. And we do think that we could use a VEGF inhibitor in combination with an EGFR inhibitor and see a greater effect. You know, great, thank you, Tonya.

Abigail: Another one for you here, do lung cells from patients express CD1d molecules?

Tonya: That's a great question. We haven't tested it in our hands, but based on the literature, yes, lung cancer cells, primary cells, do express CD1d. So, we do think that this is a great way to target lung cancer.

Abigail: That's great to know. Thank you so much, Tonya and Stacie, for answering those questions today. That's unfortunately all we've got time for, but any questions that we didn't manage to get to, we will reply via email. The webinar will be available on demand tomorrow, so look out for an email from us with the link. All that's left is to thank Tonya, and Stacy once more for a great presentation with us today, and thank you to our audience for listening. We hope you'll join us again soon.