0:58
Really appreciate it.
1:00
So as mentioned, I will be talking to you about bioassay strategy and more specifically cytotoxicity assays that you can use throughout your programme.
1:11
But before I do that, let me just very briefly introduce Abzena, which is a fully integrated partner research organisation all the way from early R&D to commercial.
1:23
And we are quite well known in the ADC space.
1:25
But what I would like to point out is that we also deal with complex biologics, monoclonal antibodies.
1:32
So it's not just ADC's, any type of biologics and we are also very scientifically driven and really focused on giving you the best state-of-the-art solutions.
1:44
We are located primarily in three sites.
1:47
So here in San Diego and Bristol, PA, we have our manufacturing sites and all the early R&D is in Cambridge, UK.
1:56
That's where my team of 25 scientists is located as well.
2:01
And most importantly, we produce results that can be seen from the multiple repeat work that we get from clients and also lots of positive feedback.
2:17
So how do we do that?
2:19
The key that we always think about while from the very beginning to the end of our programmes is derisking drug development through developability.
2:30
So what is developability?
2:31
It's basically asking 2 key questions.
2:34
Can we make it?
2:35
That is, can we scale up the manufacturing while it's still keeping the molecule stable?
2:40
And does it work?
2:41
So does it do what we expected to do based on its mode of action?
2:46
And while doing that, is it still safe?
2:51
We keep that in mind throughout the drug development process.
2:55
So in the discovery phase, we usually start with hundreds of candidates.
3:00
We run through various in silico and in vitro assays in order to narrow them down, select that one or two key lead candidates, after which we optimise and engineer them to introduce or get rid of some wanted and unwanted properties.
3:21
During the lead characterization phase, we usually have a handful of candidates that we very deeply characterise and understand.
3:29
And so by the time we go into manufacturing and satellite development with that one selected candidate, we really well understand how that functions and also the analytical characteristics.
3:45
So now moving into the main part of my talk is bio assay strategy and more specifically, how do we select appropriate assays when working with ADC programmes?
4:02
So I've selected ADCs as a mode of action because as many of our morning speakers have shown, ADCs are quite a simple concept, but quite a complex product.
4:15
And by that what I mean is there's a lot that we are asking that ADC to do.
4:19
It's required to bind to its receptor selectively, then internalise efficiently, traffic into the lysosomes, release its payload and then kill the cells through its mode of action.
4:36
How do we probe these mechanisms?
4:40
The first thing that we usually do in an ADC discovery programme, especially when we're selecting between different antibodies to use as lead, is internalisation assays.
4:54
So for this we use live cell imaging.
4:57
We use a fluorescent pH sensitive probe to label the test samples.
5:02
Test antibodies and then look at how they traffic into the lysosome and as the pH becomes lower, they light up.
5:11
So this way we get live imaging, live videos.
5:16
But because we're using a throughput, high throughput method using the Incucyte microscope, we can also quantify this and rank based on the kinetics and the dose response of internalisation.
5:31
And then once we've selected the mab to progress into becoming an ADC.
5:38
And that's where from a bioassay viewpoint, the tricky part comes because there are so many options out there to measure cytotoxicity that it can be quite overwhelming.
5:52
So that's what I aim to do during the talk is discuss with you the different options, the pros and cons and which we would recommend at what stages.
6:02
I will start by cytotoxicity assays for screening, which you would use at the beginning of the programme, and also the cytotoxicity assays for relative potency testing, which you'll be using at the end of your programme during the manufacturing phase.
6:17
And the reason I'm talking about these together is that it's usually a very similar type of assay that works well in these very early and late stages.
6:30
So the way we think about assay design and the considerations that we look into is we always make sure we have the right targets cell line.
6:41
So depending on the target and what we're trying to achieve, that can be in high, medium, low and as a control and negative.
6:50
So no expressing cell line.
6:52
We always make sure using flow cytometry that there's expression levels that are stated in the literature are actually correct and we are seeing as expected binding properties.
7:07
And then when we go into cytotoxicity, we make sure we have the right controls.
7:12
So we've had a bit of discussion about that in the morning.
7:15
We make sure that we have a relevant isotype ADC, the naked mab, free payload controls.
7:23
These are all really valuable to make sure that you are measuring the wanted effect.
7:29
And then when we go into developing the assay, we make sure that we develop to the right level that is optimised key parameters and make sure that it is reproducible.
7:45
So our workhorse cytotoxicity assay for these early and late stages is the CellTiter Glo luminous and cell viability assay.
7:52
I'm sure everyone is well familiar with that.
7:56
It's a very simple assay.
7:58
You see the cells dose and then incubate for several days and then using a luminescence plate reader, you measure viability via the ATP present at that stage.
8:17
And the really important part at this early stage is that you have reliable and reproducible data.
8:26
And how we ensure that we have that at Abzena is we do two things primarily one is data trending.
8:33
So we always run clinical benchmarks so that we know that our, we know exactly how our assay is supposed to perform.
8:41
And if something isn't working as expected, we can pick it up very early.
8:46
And we also utilise historical data so that can inform target cell line expression, for example, if target expression of the cell lines, payload sensitivity so that we make sure that we select the optimal cell line.
9:09
And now moving on a bit to the potency assays for release because as I mentioned, you would be using the same type of assay.
9:17
So they CellTiter Glo assay for that because it's simple, sensitive and robust.
9:23
But where you need to sort of ramp up the assay a bit to reach the appropriate standards is accuracy and precision.
9:34
And for that, we basically do some more assay development and qualification at this stage.
9:43
And by doing that, we make sure that the assay is performing as it is expected.
9:48
So here you can see an example of assay qualification running simulated potency samples.
9:58
And once we have that, then we're at this stage ready to do GMP ready testing for lot release.
10:05
And we have now moved into this area because we have noticed that this is a major pain point for our clients.
10:12
A lot of them say that they are able to make the assay work for lead selection, but then they're really struggling to find a partner who can actually run it to the level that is expected from the regulators for lot release.
10:32
So to summarise this part of my talk, for the early and the late stages, luminescence endpoint assays, for example, CellTiter Glo are really good solutions and are excellent options.
10:47
But what would you do in the middle of your programme?
10:50
So this is actually my favourite part, where all the weird and the wonderful happens.
10:54
And this is where you can really probe your system so that you really understand what that molecule is doing.
11:01
It's not just simply killing the cells.
11:03
What does it do in Co culture?
11:05
What does it do in 3D spheroids in various environments?
11:10
And the more information you can get, the better lead selection you can do early on.
11:18
So here the first thing that we've asked is it's good that we see what happens after 96 hours of Co-incubation, but what happens during that Co-incubation?
11:30
What's the kinetics?
11:31
How are the cells dying off?
11:34
So for this, we've ran a very similar assay as a CellTiter Glo, but in this case, we placed the plate and a real time imaging system or incucyte system and also added two dyes, the Annexin 5 Red apoptosis dye and the Cytotox Green dye for cell death.
11:56
And the example or the type of results you can get here.
12:01
As you can see immediately the cell killing using these videos, which is, which can be very powerful, especially when you're talking to investors, for example, they really like to see actually that the, that your molecule is indeed killing the cells.
12:18
But from a more scientific viewpoint, you can actually quantify that.
12:22
And in this case, we've done a cell by cell analysis, which is very similar to a flow cytometry type of analysis.
12:30
So you go through the cells as they die off, they progress from being healthy cells, double negatives into apoptotic cells, which is the red positive and then completely dead cells, which is the double positive for the red and the green dye.
12:48
And here you see examples of two HER2 ADCs and HER2 Kadcyla.
12:53
Obviously everyone uses that to generate case studies.
12:59
And what I'd like to point out is that you do see differences.
13:05
And for example, I find the apoptosis signal very intriguing because here you see a difference between the two ADCs as well as a dose dependent response in terms of where you reach the peak.
13:20
So here you can really probe differences between these two.
13:26
So then the next question is that we asked is what would happen in an environment that stimulates a solid tumour microenvironment.
13:33
So for this we've run 3D spheroid assays, in which case we use this special plate that allows spheroid formation.
13:41
And again, this is run in the incucyte.
13:44
So the nice thing about it is that you can run it to the 96 well or even more a 384 well plate format.
13:50
So you get lots of information out of it.
13:54
And the example data that you get here again using these two HER2 targeted ADCs is that these ADCs are in fact able to shrink tumours and kill the tumour cells in these 3D steroids as well.
14:11
So they are likely to be effective or they may be effective in solid tumours.
14:18
So one thing just to point out which I didn't mention that in this case the red label is not an apoptosis marker.
14:26
It's the red label is used to label the cells.
14:30
So as the tumour shrinks, the red fluorescence decreases and by the end of the time course you can see that the Kadcyla treated tumour is looking a bit miserable, less red fluorescence, whereas the trastuzumab treated one was practically growing and has still good red fluorescence.
14:59
So the next thing that we've asked is what happens in co-cultures, and in this case, think about Co cultures of target positive and negative cells, which models basically heterogeneous tumour microenvironments.
15:16
And for this we ran a bystander assay.
15:18
So just to remind everyone, the bystander effect is practically the killing of the antigen negative cells in the vicinity of the antigen positive cells.
15:30
And what our bystander assay does is if we label the positive cell line with a red dye and the negative cell line with a green dye.
15:39
And then we mix them in various ratios and add a titration of the test sample.
15:45
And through a long period of time, we monitor them using our Incucyte here.
15:51
One thing I'd like to point out that the traditional bystander assays and lots of the bystander assays that our competitors have on the market are actually not real Co culture assays.
16:03
They use supernatant transfer a lot of the times or they are endpoint assays using flow cytometry and only focusing on the target negative cell line.
16:13
Whereas in our assay with this setup, you get real co-culture, real time assessment and monitoring both cell populations at the same time.
16:27
So what you can get out of these and I'll walk you through this because there's quite a lot happening.
16:32
So if you don't treat these cells, then both cell lines proliferate.
16:37
And during the time course, you see red and green fluorescence pretty much equally.
16:43
When you treat with high concentrations of trastuzumab, you see that the red cells proliferated a bit slower.
16:50
So trastuzumab on its own is impacting a bit the HER2 positive cell line and the green cells proliferate as normal.
16:59
You treat with Kadcyla just to mention that the dosing was determined based on the single culture.
17:06
So they're determined to have maximal effect on the positive cell line and no impact on the negative cell line.
17:14
So when you treat with Kacyla, you see that the red cells die and the green cells actually proliferate even more because they spread into the space.
17:24
Whereas within HER2 you see a strikingly different effect.
17:29
The red cells die and then the green cells die.
17:32
So this is the so-called bystander effect.
17:36
And again, because we're using the incucytes, you can actually quantify this.
17:40
And now looking at Kadcyla versus in HER2, you can see that the impact on the red cells is very similar, but the effect on the green cells is strikingly different.
17:51
And that actually explains the huge difference that is seen in the clinic where in HER2 is effective in heterogeneous tumour environments where Kadcyla fails to be effective.
18:07
So now the final co-culture assay that we can do is for immune mediated killing such as ADCC.
18:18
So in case of ADCs lots of companies have the strategy to engineer out these FC mediated functions to decrease the off-site toxicities.
18:30
But some companies keep the wild typed GG1 format and aim to have both ADCC and payload mediated killing.
18:44
And just as a reminder, because these two effects occur on different time scales, it's quite easy to distinguish them.
18:52
So ADCC occurs usually in the first few hours and that we can measure by mixing PBMC effectors from healthy donors and then look at some sort of cytotoxicity readouts such as dead dye incorporation, whereas the payload mediated toxicity kills them later.
19:13
And the assay that we are currently working on and it's almost there, just not quite ready for prime time is an assay where we can actually look at the two killing effects in one assay.
19:25
So again, it will be an incucyte assay where we look at it through a longer period, and we're quite excited about that.
19:35
So to summarise this part of my talk for lead optimization and characterization, you can run lots of complex assays to really probe what your molecule is doing.
19:50
These obviously help you get a lot of information and it's helpful for better lead selection early on.
19:58
But these assays can be quite complex and sometimes do need a lot of extra effort and optimization.
20:07
So last but not least to summarise the whole talk at the beginning of the programme where you need to screen a lot of compound side by side, we recommend luminescence endpoint assays.
20:22
In the middle of your programme where you're really probing what is happening and their complex environments, you would run more complex mode of action assays.
20:32
And we're more than happy to help you with that because that can be a bit daunting if you don't have that experience.
20:39
And for supporting manufacture we and for the relative potency assays, we again recommend those luminescence endpoint assays, which again we can help you with to reach that level of regulatory requirements that are required at this stage.
20:58
And now I was focusing on ADC's, but just as I mentioned the beginning, we're not only an ADC company, we've got similar bioassay packages available for other modalities as well.
21:11
So bispecifics, AOC's, FC mediated function, practically any type of complex biologics we can assess.
21:21
So if you're interested in these, please come and talk to us.
21:24
We're in booth 20 and we also have a poster about developability, and I encourage you to visit that as well.
21:32
So last but not least, thank you very much and thank you for the great data that was provided by our bioassay team.
21:41
Thank you.