0:36
So today I'm going to talk about some work that our R&D team have been doing looking at optimising non-viral engineering of resting or naive non proliferative T cells and why I wanted to talk proliferative in a talk is beyond me.
0:56
So I'm really going to talk just a little bit about why we wanted to look at the naive non proliferative population, talk a little bit about our process that we've developed in the house for looking at delivering a number of different types of payloads for knock-out and knock-in.
1:19
The CAR-T workflow is obviously quite a complex workflow.
1:22
It's been hugely successful, but it does feature a number of different processes, many of which in the past have been open and they're quite complex and difficult from a regulatory standpoint.
1:36
Thermo Fisher has been working on a number of novel technologies.
1:41
We're interested in looking at creating modular individual units for single processes to allow more efficient scale out of manufacturing.
1:55
We're also interested in making reagents to either make the processes quicker or more effective, but also bearing in mind that everything has to have the right regulatory support.
2:06
And you know this, this workflow mostly uses viral engineering and Thermo Fisher again plays a part in that.
2:15
We have a number of tools and technologies to support viral production, but for this talk we're going to look at non-viral engineering and the products in red are the ones that are going to feature in the piece of work that our team did.
2:34
So I mentioned these closed individual automated instruments.
2:41
The two that feature in the work that we're going to talk about is the Gibco CTS Rotea, which is the counter flow centrifuge.
2:50
It allows us to do both wash and concentrate in a closed manner, but it also allows us to elutriate cells.
2:59
So we can use this to take apheresis from patients and process PBMCs from that without affinity resins.
3:09
And we're going to talk about our CTS Xenon electroporation device.
3:15
But all of these and a number of CTS, cell therapy systems is our trademark.
3:22
These indicate that either these reagents or instruments have been designed for cell and gene therapy manufacturing both from a performance point of view, but also from a regulatory point of view.
3:34
So we provide that regulatory support and documentation and also then the GMP reagents and consumables.
3:42
And we're also looking to make sure that these are all connected, or able to be connected physically using single use technologies, but digitally as well.
3:52
And we have a number of solutions for digitally connecting all of these together.
3:58
So why are we looking at non activated T cells and what are what are we actually looking at here?
4:05
These naive T cells are really the ones that are quiescent, unstimulated, haven't seen an antigen yet.
4:16
They are non-proliferating, so they're difficult to transfect.
4:21
But they do seem to have most opportunity for maintaining within patients.
4:35
And in our work, we identify the naive and stem cell memory as being the ones that are most capable of longevity and perhaps higher tumorigenicity or anti-tumorigenicity by the marker CD62L and CD45RA.
4:56
And so in the work that we're doing, we're going to be looking specifically at this set of T cell subsets.
5:05
And the reason we're looking at that is because after some of the initial CAR-T clinical trials, there's some papers published looking at the responses.
5:19
And so for a number of patients that had much better responses or less remissions, it was shown that they tended to have much higher levels of these central memory or naive T cell subsets in their starting material, indicating that that might be one of the reasons why they had a much better outcome.
5:41
So these naive T cells, as I said, they're unstimulated, they're non activating, non-dividing.
5:47
So they are difficult to transfect, but they are less prone to exhaustion potentially within therapy.
5:56
So could that give us both a safer product and give us more persistent anti-tumour effects?
6:05
We're also postulating that if you can isolate these particular cells from the patient and engineer them quickly within, within the same day or within 24 hours and do that engineering and get them back into the patient, you could have a shorter vein to vein time.
6:22
So all these things made us want to look whether we could engineer these cell types.
6:31
So as I mentioned earlier, you can either do viral engineering of T cells or non-viral.
6:38
Viral engineering is the most common at the moment, but there is a growing interest in looking at non-viral because we can give more different payloads.
6:49
So you can look at DNA, RNA or protein and you can start to look at engineering.
6:55
So actually looking at sequence specific changes as well as doing various knockouts.
7:03
And so the two systems that I'm going to talk about in this work is the research version, the Invitrogen neon NXT electroporation system.
7:12
And then it's big brother that we developed the CTS Xenon for GMP manufacture.
7:22
So a little bit more detail about these two instruments.
7:27
The Neon is a unique pipette based technology.
7:32
We've just updated it to NXT, but the system itself has been around for a number of years.
7:38
It allows you to optimise the parameters so you can change the voltage, the voltage strength, number of pulses, etcetera.
7:48
So it gives you a really nice design space to allow you to optimise your transfection conditions.
7:54
And there's a vast number of publications done on this system.
7:59
And we wanted to be able to take the way that the neon works and expand it to a GMP closed manufacturing system.
8:07
So, the Xenon uses the equivalent pulse profiles so that you can optimise on the small-scale Neon and then scale up to the Xenon.
8:17
The Xenon has a single shot cassette for one mil, but it also then has a multi shot which will do up to 2.5 billion T cells in a 25 mil volume over the course of about 25 minutes.
8:33
So these go hand in hand.
8:35
Small scale for the optimization and then, move up to the large scale for GMP manufacture as well as looking at the actual instrumentation because these non-proliferative T cells, they're very difficult to transfect.
8:55
We wanted to be able to look see if a buffer formulation might help us change the conditions that we could elect to put it under.
9:02
And so we've recently developed a low conductivity buffer that we thought would be helpful to be able to increase the voltage that we're using for these cell types.
9:14
Also to help if we have working with cell types that are prone to heat sensitivity or to allow us to work with lower concentrations of cells.
9:27
And so this has been this has been developed. Also we needed to make sure it was obviously appropriate from a regulatory point of view.
9:35
So it is a xeno-free formulation and then to support closed manufacturing as well as selling it in a bottle, we've also we're selling it off the shelf in a bag.
9:47
So that again it helps with being able to set up a closed system manufacturing more easily.
9:55
So the first step in our process, we're taking healthy apheresis from patients.
10:02
We use two donors in this set of studies and we're using the Rotea to do PBMC isolation.
10:09
So we’re really using the counter flow.
10:13
So you've got flow in one Direction and G force in the other direction.
10:17
We can separate cells by size and so we can elutriate out the platelets and plasma etcetera and we can concentrate the PBMCs.
10:28
What we're then doing is we're doing a pan T cell enrichment for these cells.
10:34
So we're using a Dynabead kit, which is the untouched human T cell kit.
10:38
And really this is just a collection of antibodies that are not CD3 antibodies.
10:44
So what's left after we remove all those others, so CD19, CD14, CD36 and CD56.
10:51
So what's left should be CD3 positive cells, but they haven't been actually touched by anything.
11:00
So before I go on to the results, just a little bit about our gating strategy.
11:06
So what we're doing is after we've identified the live cells, we're then going to be looking at CD62, CD45.
11:15
And it's this top double positive profile that we're going to be investigating for engineering efficiencies.
11:23
We're also going to look at CD25, CD69 because we're interested in these activation markers.
11:29
And so anything that moves out of this double negative space shows indication that the cells have been activated by the process.
11:43
So just pre and post T cell enrichment, we can see both from the flow graphs and then just graphically here pre and post cell isolation, we don't lose any cells in this.
11:59
But for the post-isolation we can see we have enriched for CD3 cells within this.
12:06
And then, you know, within the CD3 population, we've got the four different T cell subtypes that we're interested in including this naive and stem cell memory T cell.
12:19
This is the set of cells we're interested in looking at further.
12:26
So the rest of the protocol we're either using the Neon or the Xenon.
12:31
We transfer the cells into the low conductivity buffer for the Xenon.
12:36
There is an equivalent buffer in the Neon called T buffer.
12:41
For the gene editing, we're using our new CTS Cas9 high fidelity protein that we've recently released on the market and our TrueGuide RNAs.
12:55
We're expanding the cells with our CTS optimizer media with GlutaMAX supplementation, an immune serum replacement product and then two PeproGMP cytokines, IL-7, IL -15, because these have been shown to help with the maintenance of these naive cells without stimulating activation.
13:23
So and then we're culturing these cells post electroporation for up to 11 days and then all the data has been produced using our Attune NxT Flow Cytometer.
13:37
So the first experiments we're looking at mRNA delivery and key here, we’re just using the single shot on the Xenon.
13:47
We're just putting in a GFP using the low conductivity buffer.
13:51
And then these are the conditions that we're using.
13:53
So it's 2200 volts for 20 milliseconds.
13:56
If we were to try this with our gene editing buffers or just electroporation buffer, the Xenon would say no because it's just too high energy.
14:06
So the machine would say no because there's a high chance of arcing, but with a low conductivity buffer, we're able to do much higher levels of energy into the system.
14:18
And so again lots of data here, but essentially we've got with and without electroporation, with and without payload and in all of the different donors we're seeing that we're not losing cells.
14:29
So even though we're zapping these at quite high voltage, we're not losing cells in the process.
14:36
And for the mRNA there is a bit of donor difference, but we're getting between 60 and 80% GFP positive population within this this double positive naive stem cell population.
14:56
We also wanted to look at whether we were then activating or changing the stemness markers pre or post-electroporation.
15:08
And we don't see any change in those markers.
15:12
And again, if we're looking at the activation markers, we don't see any stimulation of activation post electroporation.
15:21
So the load, we feel this low conductivity buffer which enables that transient transfection of these quiescent or resting T cells without changing the cells phenotype.
15:32
So the next experiment we're looking at plasmid. Plasmid is a little more challenging so the same conditions at the end of it we're basically just plating out into 24 well plates and we're doing a million cells per mil in these 24 well plates.
15:51
So in this case we are again, the cells are not dying off in this process.
15:56
Transfection efficiency is a bit lower, so it's between 40 and 50%.
16:01
So obviously the mRNA was a bit more successful for actually getting expression of protein.
16:10
But again, we're able to get that transfection and transient expression with maintenance of cell viability. To start looking then at gene editing here, what we're looking to do is we're either looking at the Neon or we're looking at the Xenon.
16:33
And we're going to use a true guide against B2M to knock that out.
16:44
And again, we've worked out through a number of experiments that this the conditions at 2200 volts was the optimal.
16:55
So again, if we look at the data here again, no chain, between the xenon and the neon, we're getting sort of similar responses to the live cells, the knockout efficiency is between 15 to 20% within this population and the thing within this though we see that in these the final vials.
17:22
Remember I said that we put a million cells per mil in the cells and this is now five days later.
17:31
So the cells are not expanding, they're just maintaining.
17:33
So they're still alive.
17:35
They're not outgrowing, but really only got about 4 to 5% of the cells that are these naive T cells edited.
17:45
So clearly, if this was to be moved forward to a therapeutic application, we might need to look at post editing purification or enrichment, or perhaps even start to look at how could we then maybe activate them to expand them.
18:03
So this is by no means a complete ready to go solution.
18:10
But again, we're not seeing any change in the different T cell subtypes following the electroporation.
18:21
So our final experiment, we thought we'd do some knock-ins
18:26
So we're going to knock out the TRAC and we're going to add in a CD9 CAR into the system. Same solutions as before, either the Neon or the Xenon.
18:39
So it's final set of data then for the knockout efficiency between 20 and 40% knockout depending on the donor.
18:51
Again there is some donor variation. For the knock in it really you know, maybe 2 to 6% knock in with within the same cells.
19:01
And again it's 3 to 4% of the of the total cells in the well are edited CD64, CD48 positive cells.
19:16
And again, we're not seeing an awful lot of change in the stemness markers.
19:20
And again, post electrification we're not seeing any increase in the activation markers.
19:28
So work in progress we really wanted to prove that we could utilise this low conductivity buffer with the CTS Xenon to engineer these really difficult to engineer cells.
19:43
So it can be done and it can be done without then activating them.
19:48
The fact that with the Xenon you can optimise the different parameters of pulse, number of pulses, etcetera. It gives you a bigger design space for you to optimise your conditions.
20:02
And the increased number of buffers that we have allows you to optimise for different systems going forward.
20:14
We're looking at a Dynabead technology to be able to do purification of these post-electroporation.
20:29
And I think these will be things that will be coming out in in due course.
