0:05
So I will not be presenting any, any drug or a new therapeutic approach, but rather tools that can help for the development of new drugs and particularly for the functional evaluation of new drugs in highly customised in vitro assays.
0:18
These approaches are applicable to inflammatory context or autoimmune diseases.
0:24
But yeah, we'll focus exclusively on our immuno oncology functions basically.
0:37
So for those of you who are not yet familiar with ImmunXperts, this company was founded 10 years ago by these three people, among which Sophie Pattijn is still with the company today.
0:48
And she's very heavily involved in all our activities and she's acting as our CTO.
0:53
So you might have met her.
0:55
And over the years, the company was acquired twice.
0:57
First by Nexelis and then more recently by Q2 Solutions and these acquisitions allowed us to grow and very recently actually it was in the beginning of this year to move to this new building located in Gosselies, Belgium, which allowed us to expand our activities and also increase our staff.
1:15
Now our activities, our capabilities are subdivided into different fields.
1:20
A big part of our activities focus on unwanted immunogenicity and immunotoxicity.
1:26
Then we have our division of immuno-functionality, which covers infection, inflammation, immuno, oncology, autoimmunity, as well as cell and gene therapy.
1:36
But we also have our ImmunAcademy.
1:39
And ImmunAcademy is, for example, the possibility to develop new assays and to transfer them to your facilities if you want to establish a new assay in house.
1:48
But today I will focus only on the immuno oncology aspect of our activities.
1:54
However, one common point between all these activities is that we rely very heavily on primary human material.
2:00
So we have our own biobank with PBMC from I think it's now over 1200 donors.
2:07
And this biobank is very well characterised.
2:10
So we know the proportion of the different immune cells’ subsets.
2:14
We know the HLA type of every preparation.
2:17
We also know some of the functional aspects for every sample because we do some in vitro testing stimulating with different antigens.
2:25
So we know the responsiveness also of the different samples, which allows us sometimes to preselect donors depending on the specific needs.
2:36
Now focusing more on immuno oncology, you're all familiar with the with the immunity cycle in this context, which is very complex and there are drugs developed acting at different stages of this process.
2:48
So this is also why we are developing a large panel of different assays focusing on different immune cell subsets.
2:56
And these assays can either be used off-the-shelf.
3:00
If it's established that in a specific way and this is how you want to proceed, we can proceed like this.
3:06
But these assays can also be highly customised if your drug requires a specific context, specific additions or changes in protocols.
3:13
We are very flexible on this, but in these instances, we might need some optimizations.
3:18
And so we use a staggered approach where we work in different stages of the project with go, no go decisions.
3:24
And then after every stage we would have a call and decide on how to proceed, whether we proceed or if the protocol needs to be adapted.
3:30
And this is typically how we fine tune the assay before we actually add the final compounds in order to work with optimised conditions.
3:39
Now this is an overview of what we do in the immuno oncology team.
3:44
But I tried to classify our assays here based on the different cell types: myeloid, lymphoid cell types, et cetera.
3:50
But this is not a complete list and it's not always so easy to put one assay in one of these categories.
3:55
Sometimes they are overlapping or especially if we start mixing immune cell populations.
4:00
So I will only be able to focus on some of these assays because we don't have the time to discuss all of them.
4:06
But feel free to reach out if you have specific questions.
4:08
Also about immunogenicity aspect, my colleagues and me are here and can provide more information.
4:14
Now the first type of assay I want to discuss is the myeloid cell assay focusing on macrophages.
4:20
I believe that at this stage of the conference, macrophages do not need a long introduction anymore.
4:25
We know that both in inflammatory context and in in the context of tumours, they can have very diverse roles and come in different flavours, different phenotypes.
4:34
So historically we categorised them between these two extreme phenotypes, the M1 and M2 like phenotypes which can come in handy for the assays.
4:43
But we are not committed to this separation between these two extreme phenotypes.
4:49
Now we can use the polarisation and differentiation of these cells starting with our PBMC or monocytes isolated from our PBMC of the biobank in order to incubate them, for example, with different test products during the differentiation and polarisation context.
5:05
But also afterwards once we have generated a specific phenotype of macrophages in order to determine whether the compound can affect the polarisation of the macrophages.
5:14
So this is typically assessed by markers, but for those for those who are familiar with macrophages, they know that these markers can sometimes be also a bit conflicting, especially if you only consider M1 and M2 macrophages.
5:25
So if you see some markers being over expressed and others down regulated, sometimes you have mixed phenotypes and then the question remains, but what is eventually the function of these macrophages?
5:35
What do they do?
5:37
And to assess this we propose some functional assays.
5:41
Here you see an M2 like T cell suppression assay.
5:44
So this is an autologous setup where we isolate monocytes to generate macrophages, for instance M2 like macrophages and we can coculture them with the autologous T cells.
5:53
T cells can either be stimulated artificially with the CD3 CD28 stimulation or we can work in an MLR setup where we would have an allergenic setup to activate the T cells.
6:05
And then we can add the test compound either in this coculture already at early stages to see what the effect of the macrophages on the T cells will be, on their activation and the cytokine production, for example.
6:15
Here you see an example of such an assay where we looked at the production of interferon gamma and the proliferation of CD4 T cells which were cocultured with M1 or M2 like macrophages.
6:27
And we can see that for example, for the M2 like macrophages that there is a strong reduction of interferon gamma production as well as proliferation, indicating that they are indeed acting suppressive on the T cells.
6:38
And in such an assay, we can then add control compounds such as clinically approved antibodies targeting PD1.
6:45
But we can also add the test compounds in order to see whether we are able to remove, move or to reduce the suppressive effect of the macrophages.
6:54
In an inflammatory context, we could do the inverse.
6:56
We could try to promote inflammation, promote T cell activation and see whether the anti-inflammatory test compound could reduce this inflammation.
7:05
Another function aspect of macrophages focuses on phagocytosis.
7:10
What we do is the same procedure.
7:12
We assayed monocytes generate the macrophages which can be co-incubated for example with antibodies targeting a specific type of cell or we could also incubate them with fluorescently labelled bacteria or beads depending on what the compound is actually targeting, more the macrophages or more target cell line.
7:29
And then we can perform different types of read out endpoint analysis by flow cytometry or live cell imaging.
7:34
If you are not entirely sure about the kinetics, you'll see an example of a flow cytometry readout where we had a B cell lymphoma cell line which was incubated with either Rituximab or the IgG1 isotype control.
7:50
Until you can see that by flow cytometry analysis, we are able to detect the uptake of the cells, or the phagocytosis because these cells were pre-labelled with a proliferation dye.
7:59
So we can look into the macrophages and see whether they perform phagocytosis or not.
8:05
The other approach is the live cell imaging based approach, where here we label the cancer cells with a pH sensitive dye and when the cells phagocytose, we can see over time an increase of the fluorescence intensity and thereby track phagocytosis, which can come in handy if we are not entirely sure about the kinetics.
8:24
Another type of cell for which you perform functions are natural killer cells.
8:27
So natural killer cells, they can also have diverse roles, but they are very famous for antibody dependent cellular cytotoxicity.
8:34
So in this context we would for example test whether an antibody can trigger an ADCC response.
8:41
So we have the engagement of the FC gamma receptor on the natural killer cells which could bind to the Fc fraction of the of the antibodies if it binds to the target cell line.
8:50
And this can be assessed by isolating NK cells from our PBMC and coculturing them with target cells in the presence of the test compound.
8:58
And then we can again perform different types of readout of either live cell imaging based, or flow cytometry based depending on the on the setup.
9:07
An example of this is flow cytometry based where we co cultured NK cells with A549 cells which were treated either with Cetuximab or the isotype control and then performing flow cytometry readout we can look at the degranulation of natural killer cells and quantify the data.
9:31
So regarding a lifestyle imaging based readout the setup would be slightly different.
9:35
We coculture them with the test compound and in this case the target cells are prelabelled with a fluorescent dye.
9:41
This allows us to track the number of target cells over time, but we also add the cytotoxicity dye in order to detect cytotoxicity events and then track all this over time.
9:52
The double readout can have an advantage because some cells are more easily identifiable than other ones.
10:00
So using this double readout it can cover, or it can provide additional chances to actually track reliably cytotoxicity over time.
10:13
Here you see an example because this kind of killing assay can not only be performed with NK cells, but also with T cells with PBMC.
10:19
And this is an example of PBMC.
10:21
You can see this double readout tracking the amount of target cells over time, how it can reduce when we add, for example, a test compound or stimulate the cells or look at cytotoxicity and see how it increases over time.
10:35
And this is what provides us this double readout.
10:37
And this is in the inter side system tracked over time, which can provide some nice movies, but it's not always easy to visualise because sometimes we need quite a high effector to target ratio, especially working with cell lines like A549 cells.
10:50
So it looks a bit crowded, but we can clearly see the red events, the cytotoxic events in the cytotoxicity map condition versus the isotype control.
10:58
Now regarding T cell assays, T cells are very important in the context of cancer.
11:03
This we are familiar with, but actually when we want to set up an assay there are different setups that are possible.
11:08
We can either trigger antigen specific activation of T cells for example by isolating CMV responsive donors or priming cells to have an enrichment of cells specific to a MART-1 peptide for example.
11:23
But we can also work in an allergenic activation.
11:25
So for example, the typical mixed lymphocyte reaction where you have an incompatibility which will we got T cell activation and then assess the effect of a compound in this setup.
11:35
And finally the last setup is the nonspecific activation of T cells with either superantigens or CD3/CD28 stimulation as I presented previously.
11:45
Now this is an example of a CMV reactivation assay.
11:48
So we had from our biobank selected the donors, we stimulated the T cells with the specific antigen and added the test compounds and then we can see whether the test compounds will enhance for example pre inflammatory cytokine secretion or T cell proliferation.
12:06
Here you see an example of such an assay where we have CD4 T cells and after 5 days of incubation we looked and the proliferation of the T cells as well as the interferon gamma secretion and we can see that there is an increase when we when we stimulate the cells with the peptide stimulation which can be further enhanced when we add an anti PD1 antibody.
12:28
In this condition, always use the controlled antibodies for reference and then we can also test additional test compounds to see whether they are also able to promote the T cell activation, the proliferation or the cytokine production.
12:43
Now these cocultures can also become a bit more complex.
12:46
For example, we can add another cell type in there, for example, suppressive cells such as Tregs or M2, like macrophages.
12:53
And what you see here is an example where we had pre-sorted for Tregs and these Tregs were added in an MLR setup.
13:00
So in the MLR, we track T cell proliferation over time where we did see some proliferation which was reduced when we added the suppressive cell type in the in the condition.
13:09
And then when we add the test compound such as for example, the anti-PD1 immune checkpoint blockade, there we see that we are partially able to restore the proliferation or again promote T cell proliferation.
13:20
And in such a setup we could test a wide range of compounds and always compare to reference molecules.
13:28
Now an even more complex setup is the DC-T assay.
13:31
But this is something which allows us to test compounds which target different steps of the priming of the effect of phase.
13:41
What we do here is in this autologous setup generate monocytes which we differentiated, which we differentiate into immature dendritic cells and these ones are done maturated.
13:54
And during this maturation process, we can for example, electroporate them to express some mRNA if you want to test a vaccine for example.
14:02
Or we can add other compounds to see whether they interfere in the priming process or simply load mature dendritic cells with a peptide.
14:10
These mature dendritic cells or loaded cells are then co-incubated with the autologous T cells in order to induce the priming.
14:18
After the priming we can assess T cell reactivity by re exposing them to the antigen either by loading monocytes with the with the peptide for example and then co-incubating these two types of cells.
14:29
And then we have different readouts possible.
14:31
We can on one hand perform a tetramer staining to see the percentage of reactivity.
14:35
We can also perform FluoroSpot or intracellular cytokine staining in order to see T cell responses to a specific antigen.
14:47
Now there are additional assays available but due to time constraints I cannot explain all of them.
14:52
But just to give you a glimpse of what is also possible and what you can further discuss after the meeting is for instance an exhaustion assay but also a CDC assay.
15:01
So complement dependent cytotoxicity assay which we recently developed.
15:06
Give an example of primary B cells or can also use it with B cell lines and where we can track over time in a live cell imaging based approach complement mediated cytotoxicity.
15:17
And finally, this is more linking to immunogenicity, a MAPPS assay of immunopeptidomics where we could check whether a specific antigen can be presented by the cells.
15:28
And this is done in collaboration with a company called ImmuneSpec, who will do the mass spec part, and we will do the cellular part of the validation steps afterwards.
15:39
Now, I mentioned that we have a large panel of assets which is available.
15:43
I couldn't mention all of them today.
15:45
But what I wanted to stress is that most of our assays rely on human material from our biobank or from fresh blood.
15:51
But we also perform several of these assays with cyno material or with murine material.
15:56
This is rarer, but for cyno assays, there is also an increasing demand.
16:01
So if you have additional question on those assays, feel free to approach us.
16:07
And what I presented here is of course not all my work.
16:10
I joined the company only a few months ago.
16:11
So there is a whole team.
16:12
We are now, I believe 32 people located in the brand new lab in Belgium.
