Beckman Coulter Life Sciences
Duration: 12:48 Min
Cell and Gene Therapy Biomarkers
Transcript
0:01
Absolutely. Great talk, Vasundhara. Thanks a lot for the segue. I mean,
0:09
it was very interesting looking at what you were doing. And as you said that we are also having
0:14
some other molecular techniques which can actually complement the data which is produced
0:20
by CellDive and actually overlay over the spatial information that is coming there
0:29
and add more substance to the biomarkers. So I'm coming from Beckman Coulter Life Sciences,
0:40
and we are actually looking at certain molecular biomarkers that can be
0:46
used specifically for the cell and gene therapy workflow.
0:51
So coming to the cell, can you go to the next slide, please?
0:56
Yeah, so there's a very rudimentary overview of the cell and gene therapy
1:04
workflow where you have one part is the production of the cell and gene therapy
1:12
products that is either vectors or cells or different kinds of modifications which are
1:19
made on the cells. And the next part is going towards the clinical trials where you're actually
1:25
testing these things in patients, in animals to actually see the efficacy of these products.
1:35
So a biomarker effectively or a nice biomarker would be that would be able to,
1:42
you will be able to track the whole process starting from production where you will,
1:48
I mean, you would want the quantification of viral copy numbers, detecting gene editing or
1:53
reprogramming viral vectors, going to QC impurity testing where you would need the copy number
2:02
quantification, mycoplasma detection, off-target effect detection, going towards clinical trials
2:09
where the biomarkers can be directly used for, you know, looking at the progression of the disease or
2:15
how well the, how effective the therapy has been, and also the persistence of the transformed cells
2:22
to have a better idea of the actual working of the system. So could go to the next slide please.
2:30
Yeah, so what, how should this biomarker look like? So I would say a type of biomarker would
2:41
be a small molecule, immunological or molecular. So you could have different types of these kind
2:46
of things. The analytical testing method is very important for the biomarker because
2:55
that is going to be important for the safety and efficacy testing of these therapies.
3:02
For clinical trials, we would want the biomarker to monitor the treatment dose, target cell
3:08
population, and other indicators for safety and efficacy. And the other biomarkers could be also
3:15
looking at the immune fitness of the cell or of the organism or human beings to see the
3:23
monitor the impact of these therapies. And we have different types of products like you have,
3:29
you know, autologous and allogeneic CAR-T, and these can also impact the analytical and
3:35
biomarker testing. We have various parameters to consider when you're actually looking at
3:41
an effective biomarker, which would be able to accomplish a lot of things in the organism.
3:49
But also, if the biomarker were to be useful upstream during the production, that would really
3:54
make it a seamless translation from the production to the clinical trials and to the organism.
4:02
To go to the next slide, please. Thank you. Yeah, so when we are, what are the different
4:08
technologies which we are using for biomarker testing in clinical trials? So, as you see,
4:13
this is from Nature publications, and, you know, there are various technologies, so I would not go
4:20
deep into it. So, Vasundhara had presented the fluorescence immunoassay, microscopy,
4:27
spatial imaging, so that's what is marked in green, and also microscopy and immunohistochemistry. So,
4:37
that is all the technology which is coming from Leica. And from Beckman Coulter Life Sciences,
4:43
we have this, our core business, which is the, what you call, the immune monitoring and looking
4:49
at the immune fitness of the organisms. So, and what I'm going to be presenting today would be
4:55
the liquid biopsy and the DNA sequencing part of it. So, we are also developing
5:00
clinical trials and to the organism. To go to the next slide, please, thank you.
5:06
Yeah, so when we are, what are the different technologies which we are using for biomarker testing in clinical trials?
5:13
So, as you see this from this from Nature publications and you know there are various technologies, so I would not go deep into it.
5:23
Thank you.
5:24
So that is all the technology which is coming from Leica and from Beckman Coulter Life Sciences. We have this our core business, which is the what you call the immune monitoring and looking at the fitness of the organisms.
5:51
So and what I'm going to be presenting today would be the liquid biopsy and the DNA sequencing part of it.
5:58
So we are also developing new molecular techniques to complement our flow cytometry data, which is which is which is talking about the immune status of the patient.
6:11
And what we are proposing with our liquid biopsy and our amplification protocols is that we can track specific sequences and tumor-specific mutations as biomarkers.
6:25
So the current methodology of choice is next-generation sequencing.
6:32
It generates enormous sequence data to show where the mutations are present.
6:38
It's a very powerful technique, but unfortunately, it is also very cumbersome to do the long turnaround time, three to six weeks, and also very expensive to do it multiple times.
6:52
You do need the next-generation sequencing to produce the markers.
6:56
But to track these markers and track them through the production cycle and to the clinical trials would be a very expensive and cumbersome process to follow.
7:07
So what we are proposing are these liquid biopsy techniques where we could detect DNA mutations and DNA sequences through the cell-free DNA, which is present in the plasma of the blood.
7:24
So instead of having to do biopsy of the tumor tissue and repeated invasive procedures, we can actually measure the same biomarker in blood, which makes it very easy for access of sample and also has a very minimal impact on the patients.
7:46
And as I said, you need prior sequence information for this assay, which is provided by the NGS.
7:51
And to complement it, we have the deep immunophenotyping by flow cytometry, which gives you a very clear picture of the immune system, how it is reacting to these therapies and a reliable assessment of the immune function.
8:08
Go to the next slide.
8:11
So I would quickly go through the technology, what we are talking about.
8:14
So it's a linear amplification technology, which is called rolling circle amplification instead of the PCR.
8:23
The PCR has the capability of generating huge amounts of amplification.
8:28
But unfortunately, due to the errors which are caused by the polymerase, you have a lot of information.
8:36
So you have high sensitivity, but low specificity.
8:41
But by using this rolling circle amplification, you are able to generate huge amounts of amplification with practically minimal, with very minimal errors in it.
8:52
And so what happens in this technology, you extract the DNA from blood, the normal procedure.
9:00
Then you have these padlock probes which bind to the region of interest of the DNA.
9:06
It amplifies it quickly by rolling circle amplification.
9:12
And then you put the genotyping probes specific to the wild-type and the mutant DNA.
9:18
And then you perform the rolling circle amplification yet again.
9:22
So you're able to generate very specific sequencing information, very specific mutation information from this kind of amplification.
9:31
And the final DNA molecule actually becomes into a popcorn-like structure that you see on the right-hand side of the screen.
9:38
And this DNA structure, which is actually fluorescent because of the fluorescent-linked probe, can now be tracked through flow cytometry.
9:46
So in the lower right-hand side plot that you see is at the bottom, you have the wild-type.
9:53
And on the left-hand side vertical scale, you have the mutant DNA, mutant particles.
10:03
So you can very easily calculate the mutant allele frequency or whatever.
10:07
So it's a very easy way of tracking mutations, which can be very powerful biomarkers.
10:13
But there's an amplification technique. So you can also amplify sequences.
10:20
So when you have CAR T cells which are producing or you make these CRISPR mutations,
10:26
then you can actually track these sequences of CAR T in your blood for a long time.
10:33
So you can actually look at the persistence of the CAR T cells, persistence of these CRISPR modified cells or vector modified cells.
10:42
So it's an amplification technique which has multiple applications, starting from mutation detection to a general amplification.
10:52
So you can see that it can actually be used across right starting from your production to your clinical trials.
11:03
Go to the next slide, please.
11:07
So, as I said, the RCA assay is highly sensitive. It's got 10 to 100 fold higher sensitivity than ddPCR or NGS.
11:17
So the publications have actually shown this thing.
11:20
And then because of the high number of events that you're acquiring through flow cytometry.
11:27
And you don't need any specialized equipment except a flow cytometer, which you would have along with for your immunophenotyping anyhow.
11:35
It's high throughput. It can be multiplexed and it's easy to set up in a lab.
11:41
Practically takes a day to set up the whole assay. And then you can do it.
11:45
And it's a far lower cost than NGS would say.
11:50
So different applications that you can actually look for are mutation detection in diseases, persistence of transformed cells,
11:58
copy number quantification and can also be applied for mycoplasma detection, contamination detection, purity checks of target effects.
12:05
So it's a whole plethora of amplification techniques that can be used or biomarkers that can be identified using this technique.
12:16
And as we saw from the previous thing, this information can also be overlaid over the data.
12:22
This is coming from proteomics, from imaging and other techniques to give a very powerful system-wide approach,
12:30
knowledge of what is actually happening due to these therapies that we are trying out.
12:36
So that's all I have to present. And then I would thank you for your time and attention and your questions for the both of us.
12:44
Thanks. Thank you. Thank you.
12:48
Yeah, thank you, Shankar, as well. If there's any questions from anybody online or anybody in the room, we'd be happy to take any questions.