Hello, everyone, and welcome to today’s live broadcast, The Inhibitory Role of Specific Macrophages in RCC.

I’m Alexis Kraus of Laberits, and I’ll be your moderator for today’s event.

Today’s educational web seminar is brought to you by Laberits and sponsored by Visiopharm.

For more information about our sponsor, please visit their website at Visiopharm.com. Now let’s get started.

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I will now turn the presentation over to Brandon Hopkinson to introduce our speaker for today. Brandon, welcome, sir.

Hello, and thanks for joining us today. My name is Brandon Hopkinson. I’m a product marketing manager here at Visiopharm, and I’ll be your moderator for this lecture.

Today, we have doctor Alfreda Nersnel, head of the immunoanalytics research group at the Helmholtz Institute and a professor at the Lundbeck Maximilianus University in Munich. She is a board certified immunologist with special expertise in antigen presentation and T cell modulation.

During today’s webinar, she will discuss the inhibitory role of macrophages in immuno oncology with a focus on renal cell carcinoma.

Thank you very much for your time today, doctor Nuschno. I’m excited for the presentation, so I’ll let you take it from here. Please begin whenever you’re ready.

Thank you very much for this very nice introduction, and I welcome you all to today’s webinar.

And the title is the inhibitory role of macrophages in in immuno oncology and laryngocyte carcinoma.

I will start with the current status of immunotherapy or cancer oncology therapy, and I introduce you to the new area of immuno oncology.

This is with two new therapies, the adoptive T cell transfer and the checkpoint inhibition. Here shown the checkpoint inhibition. And what is new about it and what’s the new area about is that now we can achieve long term control of tumors even in metastatic situation as you can see in the top. So for patients who have failed all therapies, now there are options to to to achieve long term remission and durable responses.

And here shown in green is the survival curve of ipilimumab, and in blue, the nivolumab monotherapies to checkpoint inhibitors.

The immunotherapy started a new paradigm in tumor therapy. It’s no longer like with current or classical cancer therapies that the tumor tell cells are the direct target of therapeutic inhibition, like chemotherapy, tyrosine kinase inhibitors, or anti BGF therapy, they target the tumor cells directly and kill them. However, tumor cells who do not have the target molecule, they are resistant, and the tumors will eventually grow out.

Immunotherapy is we mobilize our own defense system to fight the tumor cell. As we do all know, is the immune system is dysfunctional in the tumor microenvironment and the new therapeutic applications reactivate those existing t cells by releasing the break with anti CTLA-4 or anti PD-1, PD-L1 checkpoint antibodies.

The consequence is that the t cells will become reactivated and now can kill and destroy the tumor cells. The second therapy is adaptive t cell transfer of engineered t cells where the t cells are equipped with new anti with new receptors, like the chimeric antigen receptors, which we know as the c d nineteen, CAR T cells for the treatment of leukemia and lymphoma.

Here, new t cells are given to the patient, and they can direct directly attack and destroy the tumor cells.

This new therapy was possible through scientific knowledge by two seminal research scientists, Jim Allison and Tasuku Honjo, who discovered Jim Allison discovered the CTLA-4 in 1987, and he recognized that by inhibiting the interaction of CTLA-4 with with its ligand can initiate an immune response. Harnio discovered the PD-1 and discovered or recognized that this is a negative regulator of immune response.

And the scientific knowledge was translated to clinical application by the development of a number of different monoclonal antibodies, nivolumab, pembrolizumab, atezolizumab, divalumab, ipilimumab, and so on. There is now a number of therapeutic antibodies which are active and effective in clinical application.

So the mode of action is that, like, anti PD-1 or anti PD-L1, they release the break in the effector phase, so in the tumor microenvironment with the consequence that more tumor cells are killed with existing specificity.

Anti CTLA-4 works in the priming in the initiation phase of the immune response. It takes out the break in the priming phase, leading that more t cells and t cells with new specificities are being generated.

The advantage of the new specificities is that now additional tumor cells can be killed. However, that has also the risk of autoimmunity because maybe, possibly, also self cells are being killed.

The current status with this new development is that we can achieve long term tumor control in a number of tumor entities. We have those tumors that respond very strongly, like Hodgkin’s disease or Merkel cell carcinoma.

We have most tumors who respond immediately, like melanoma, lung cancer, bladder cancer, renal cell cancer, and so on. So it has to be noted that we have responses across many tumor types. However, there are some tumors like pancreatic or prostate who do not respond or do not respond very well. In general, we have to say that only a subgroup of patient responds. And the question is, who is a responder?

Why are others non responders? And how can we turn a non responder to into a responder?

Currently, experiments are done with combination therapies here shown anti CTLA-4 and anti PD-L1. And you can see the response rates are increased with regards to comparison ipilimumab and nivolumab here in green and blue, compared to the combination treatment with nivolumab plus ipilimumab in yellow. However, still, we have forty percent non responders.

And I’ll I’ll highlight here that we have a combination of two t cell targeting therapies.

What I want to show you in the next slide that the immune system is much more complex than just having t cells to do the job. We need to know what the immune cells are required, what they do need for rejection. And we have to find out case individually, meaning personalized therapy, where is the problem. For that, we have to look at the tissue environment and see actually what the current status of the immune system is at the tissue site.

And how can we do that? For that, we have to look at the tissue. We have to characterize the tissue environment, and we have to find out not only the number, but also the type and the functional orientation of the immune system. We have to find out about the localization of the immune cells.

Are they in the tumor core, in the invasive margin, in the stroma, or even are they in the metastatic site or in the primary tumor?

And finally, we also have to find out whether the immune cells communicate with each other and how and what they communicate about.

How can we do that? How can we define the immune contexture?

Those of type, density, quality, localization, communication.

We have we can take the tissue, dissociated, and acquire the Till, so the tumor infiltrating immune cells, and do a flow cytometry analysis.

By that, we can find out what type of t cell is what type of immune cell is there. And as an example, for the renal cell cancer, we have about a one to one ratio of t cells and myeloid cells. So it’s not only t cells, which are in the tissue present. Then we have a small number of NK cells, meaning natural killer cells and b cells.

With this type of analysis, we can have a deep insight into how and what type of immune cell are there, but what we do not gain is where are they located and do they communicate.

For that, we have to look at the tissue. We have to look into the tissue site where actually the communication might take place. And by that, we can see that as an example, the renal cell carcinoma, we have t cells here stained in red, and k cells, again stained in red, but we also have a number of myeloid cells by different markers, and we can also see that they do have different localization.

For example, the DC lam positive lymphocytes, leukocytes are focially oriented, wherein the other ones are dispersed.

The different immune cell types, they also do have different functional consequences and importances with regards to the arrival. Here, we’re looking at, colorectal cancer, CRC, or immuno cell cancer tissue.

We look at the CD eight t cells, and we can see here that the number of the CD eight t cells in the colorectal cancer has a positive, predictive value for survival, while for renal cell cancer, it has a negative predictive value. The curves always are the ones in red. And k cells, for example, quite differently, have a positive predictive value in renal cell cancer and have no value in colorectal cancer. The d z lam positive till, again, in the colorectal cancer, they have a positive predictive value. In the renal cell cancer, they have a negative predictive value. So the different immune cell types, they can have different prognostic value depending on the tissue type.

And how can we further use this knowledge for the benefit of the patient?

So we, in our lab, we looked at a different type of myeloid cell that expresses the marker DC sign. This is a marker for interstitial, so tissue resident myeloid cells as we can find them in the tumor tissue.

We observed that myeloid cells with the CD two zero nine surface marker, which we call ERP DC, because they are particularly frequent in renal cell carcinoma, ERP meaning enriched in renal cell carcinoma.

And we observed that a high number of myeloid cells with this marker, CD two zero nine, indicates a poor prognosis. So the curve in red, high ERTC number, meaning poor survival probability.

While patients with tumors with low ERTC numbers have a better survival probability.

And with regards to, therapeutic consequences, we have to consider what the mode of action is of these myeloid cells. Why are they contributing to a poor survival? Is it because they are directly tumor promoting by secreting factors like VEGF, TGF beta, IL ten, or inhibitory surface molecules?

Or is it because they communicate with other cells, like a t cell?

And as an example, they might have surface molecules that are inhibitory to t cells, so preventing the lytic activity of the t cell, destroying the tumor cell. And that has a therapeutic consequence. If we consider PD-1, PD-1 checkpoint inhibition therapy, the so this interaction of PD-1 with PD-L1 is also a negative signal. And by checkpoint antibodies, we take out this negative signal.

If we have macrophages providing also a negative signal, this will prevail even in the presence of checkpoint inhibition therapy.

Meaning, we have resistance to checkpoint therapy because the macrophages still are inhibitory.

So we have to find out, is there indeed a communication between these two cell types in the tissue?

And we looked adrenal cell carcinoma tissue by chromogenic, double immunohistology, like CD3 in blue and CD2 zero nine in brown, so our myeloid cells. And we do see that these two cell types are indeed very adjacent to each other. So we can assume that they communicate, and we would like to know actually what do they communicate.

And for that, we need to stay in a few more markers.

And for that, we do need to get into a more multiplex immuno histology with using fluorescently labeled antibodies.

And this IF, so immunofluorescent immunohistology provides the opportunity to use more than two markers. And like here, we stained CD three for the t cell in blue, the macrophage in green, and a third marker, FOXO, which is a transcription factor.

And this transcription factor is known to provide cell cycle arrest. It is also known to drive the t cells away from effector function, so inhibiting their killing activity.

And when you look now at this a multiple stained immunofluorescently stained tissue section, we find a situation where a T cell is adjacent to the macrophage and another t cell is not adjacent to a macrophage. And if we look at these two different t cells for the expression of the transcription factor FOXO, we can see that FOXO is expressed in the t cell that is adjacent to a macrophage, and it’s not expressed in the t cell that is not adjacent to a macrophage. And this information provides us with the knowledge that, indeed, the macrophage may communicate something negatively to the t cell and potentially inhibiting the t cell.

So we can assume that the tumor associated macrophages are indeed inhibitory cells in the tumor microenvironment, and by that, they are relevant targets in the oncology.

There’s a number of publications that indicate this concept that the macrophages may limit chemotherapy, it may limit immunotherapy, and that they are by that interesting targets in oncology.

If we know that they communicate with T cells, we do also know that a therapy that is directed towards activating the T cells cannot by itself be optimally active.

Therefore, targeting strategies to remove, to change, to eliminate macrophages may be be beneficially combined with checkpoint inhibition.

Again, multiplex phenotyping of the tissue provides the relevant information whether or not we have to do combination therapy and what combination therapy might be the most effective. Here shown again an example of the renal cell cancer where we have on the top a type of renal cell cancer that has high macrophage numbers, like here, the cells in red, and in green, it’s the t cells. So on the bottom, we have an example also of ring’s leg cancer that seem to have very little macrophages present.

We can assume from this knowledge that the top patient with a tumor that has macrophages, we likely expect resistance to checkpoint monotherapy.

The bottom patient with a tumor that has very little macrophages, we expect that it’s likely that this patient is responsive to monotherapy of checkpoint inhibition.

So it has a consequence what type of immune cells we have present in the tissue type. In addition, it is important to solve and to recognize the spatial resolution.

So whether the immune cells are directly adjacent to the tumor, like here intraepithelial, here, this lymphocyte is directly in the tumor nest, whereas all the other lymphocytes here are in the tumor stroma, so not really directly in contact to the tumor cell.

Also, with tissue phenotyping, we can determine whether we have communication, like in this situation where the suppressor in red is distance to the infector in green. So here we would assume communication does not necessarily take place, and we do not have to consider very strongly in therapy the suppressants of the suppressor cell. Quite differently to this tumor type, where the suppressor in red is directly adjacent to the effector in green, where we do expect that we have to target both cell types to get a beneficial therapeutic outcome. And finally, we do not want to only look at a very tiny slice of the tissue because the adjacent tissue may be different.

So we want to scan the whole type the whole section of the tissue to really completely harvest the information that the tissue provides us. And you will recognize immediately, now we really have to get digital. We have to have a good screening system, a computer assisted digital observation of the tissue to really harvest everything that the tissue tells us for the benefit of the patient.

This is my last slide. I thank you for your attention.

I hope I could communicate to you the importance to find out the complexity of the immune system in the tissue type, and I hope I could also show you that although we all believe that our eyes are the best evaluation system, we do need in the future to look broader and to help our own personal, evaluation by computer assisted, technologies. Thank you for attention.

Great. Thank you very much for the excellent presentation.

We’re using digital image analysis, especially for those complex immuno oncology questions, can really detail out the players in those systems.

We would like to thank both doctor Nirschnow for her time as well as everyone who watched today’s presentation.

If you have any questions, please feel free to contact us at webinars@visiopharm.com or check out our other webinars or additional content available in the knowledge library on the Visiopharm website.

Thanks again, and have a great day.

Thank you, Brandon. And we would like to once again thank our audience for joining us today and for their interesting questions.

All questions asked will be addressed by our speaker via the email address that you provided at the time of registration.

We would like to thank Laberits and our sponsor, Visiopharm, for underwriting today’s educational webcast.

You can view this webinar on demand. Laberits will alert you via email when it’s available for replay.

Be sure to join us for our next webinar sponsored by Visiopharm, development and analytical validation of prognostic biomarkers for metastasis on February ninth at nine AM Pacific, twelve PM eastern.

That’s all for now. Until next time. Thanks for joining us. Bye bye.