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I'm going to talk a little bit about animal models and I'm glad to have the opportunity to share information about animal model application in anybody discovery with you.
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Here is the outline of my talk and here is a little bit of background information.
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Since the first anybody received approval in 1986, we've seen an impressive surge in development and approval of antibody drugs by 2021.
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In April 1,100 antibodies got approved illustrating the rapidly expanding of the market and this growth reflects increasing needs for antibody based solutions across various of diseases.
1:10
And the antibodies are also the structure base of many different kind of drugs and the antibody plus conception was raised in 2022 by scientists.
1:27
The antibody plus drugs incorporate the effector modules ranging from small molecules, nuclear acids, peptides, proteins and even cellules to enhance their effectiveness.
1:44
Indeed, advancement in technology had been had significantly accelerated the pace of development and application of antibody drugs.
1:58
But still there are challenges and uncertainties in this field.
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The investment of time and cost in this field is substantial, but still with very high rate of failure and challenges raise up in almost every aspect of the development of drugs like target selection, protein engineering, CMC process and evaluation system.
2:34
And I'm going to talk about the immunogenicity as an example, the low human components in the antibody drugs maybe immunogenic potentially leading to failure through induction of ADA and generally the higher degree of humanization, the lower the risk of immunogenicity.
3:04
And but as we know a predicting the immunogenicity risk in pre-clinical studies is very difficult.
3:13
Therefore antibody drugs, especially those use in long term face significant uncertainty.
3:24
And due to, I have an example about that the Bococizumab was terminated in phase three because of the immunogenicity issue.
3:39
And I want to solve this problem we need to use the humanised antibody or fully human antibody thereby reduce the potential risks, risks in later development stage.
4:00
And when we talk about the antibody discovery, we are referring to technologies like a hybridoma phage display, single B screening or use of a transgenic model.
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And typically we start from mice, we immunise the mice and collect splenocyte to create hybridoma which is essentially a factory of antibodies we are interested in.
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But there is a twist, those antibodies are non human and need to undergo humanization engineering the antibodies to be more human like and reducing the risk of being recognised by the human's immune system.
4:48
Well, phage display provides a rapid approach to screen antibodies from large libraries.
4:56
However, we should consider about the concern about the problems lead by mismatches of VH and VL.
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On the other hand, single B screening technology allows us to screen human B cells directly, but the affinity of the antibodies are relatively low.
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In most cases.
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When transgenic mice are used for immunisation, the antibodies are encoded directly with human genes and the B cells are developed in natural endogenous immune system and thereby produce higher quality of antibodies.
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This not only save us tremendous of time and money by skipping the humanization step, but also significantly lowers the risks of immunogenicity and maybe other part of risks.
6:04
And what's more, these transgenic mice are universal feat for all the technologies we've talked about.
6:14
I think This is why transgender mouses will come in this field.
6:24
Well, when it comes to choosing mice for immunisation for discovery or antibodies, above you see mice almost universally selected.
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Why is this so?
6:40
The answer lies in their performance in in generating a robust humour humoral immune response.
6:49
And when we'll take a closer look at the MHC 2 alleles across various of inbred mouth strains, BALB/c mice clearly stand out.
7:02
As you can see, their MHC genes are fully functional, contrasting to other inbred strains with at least one allele defective.
7:14
And the distinction is particularly noteworthy when we look at the MHC 2 alleless, which plays a critical role in CD4T cell development and activation, which is essential factor in effectiveness of humoral immune response.
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To illustrate this point, let me share a published case study published in 2022.
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The authors immunised different groups of mice with influenza peptide vaccine and as we can see the BALB/c mice equipped with 40 functional MHC 2 I-E and I-E molecules show the robust immune response against the immunisation.
8:16
Well.
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Look at the B6 mice lacking the functional I-E.
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MHC2 molecules had a significantly weak response and this example underscores the Super immunological capabilities of BALB/c strain.
8:40
Building on the BALB/c impressive attributes, we've developed the new map model which is a 40 antibody transgenic mouse model directly in BALB/c background.
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We directed the ES cell line from BALB/c strain and introduced megabase sized human fragment contain all the human VGJ genes into the mouse chromosome.
9:10
And essentially we've replaced the mouse variable regions for heavy chain and Kappa light chain with human genes while retaining the constant region gene intact.
9:25
And this design ensures the FC part of the immunoglobulins can interact with the FC receptors expressed on many different immune cells and enable the standard immune system development and response normally.
9:48
Also, we created three different version of NeoMab mice tailored to different research needs.
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The standard IgG version is established for a whole IgG discovery and the heavy chain only model for VHH antibody discovery also a common light chain model for best specific antibody development now.
10:20
And each model is designed to harness the full potential of human antibody discovery in mouse model.
10:28
And all the models are established based on the NeoMab.
10:36
Based on this one, to assess the effecting effectiveness of NeoMab we conducted a lot of evaluations including the human gene usage, immune profiling and B cell development.
11:02
And as you can, as you can see in this slide, look at the left figure, you'll see all the human genes we put into the mouse.
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They are used by the animal model to increase to encode the immunoglobulins just like in humans.
11:24
And what's more, the genes are used with their frequency that's very much in line with they are used in humans, which means the higher frequency used preferred VH genes or VL genes in this model is also used like the or they are used to encode the immunoglobulins in humans.
11:56
This means that the human gene usage in NeoMab mice closely mirrors the use in in human both in terms of genes and their frequency and look at this figure.
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The sequencing diversity index is similar between NeoMab and human both heavy chain and light chain.
12:19
This is important because it suggested that the antibodies produced by NeoMab is very human like.
12:30
And let's look at the specific part of the antibody CDR3H which is varies in lengths between species.
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On average CDR3H is a little bit longer in human than mouse.
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And as you can see, CDR3H lengths of NeoMab is quite similar to that of human, but significantly different from its background strain BALB/c ok, which means the structure of the antibody produced the NeoMab is highly human like.
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We also examine the immune cell proportion in both spleen and blood, and NeoMab and BALB/c seem pretty much the same, suggesting the immune system is competent and functional.
13:30
And also we looked at the development of B cell and the proportion of B cell at different developmental stage is similar between BALB/c and the NeoMab.
13:45
We also observed all the different types of immunoglobulins in the server of NeoMab both before and after immunisation and the level of the immunoglobulins are quite similar between NeoMab and its background strain.
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BALB/c, Understanding the strong immune response is a key for obtaining high quality antibodies.
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So we put the NeoMabs to the test against a wide range of antigens including cytokines, peptides, soluble proteins and GPCRS will also test their case or try a case using the mRNA as antigen.
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And as you can see in this slide, overall NeoMabs can respond very well against the different kind of antigen immunisation.
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And in some cases, NeoMabs respond much better than its background stream, BALB/c.
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And we also see the much better immunisation against the mRNA immunisation in this figure.
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After the first dose, NeoMabs can respond very well, while BALB/c have nearly no respond at all.
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And to evaluate how well the NeoMab model works for discovering antibodies, we also carried out several experiments targeting different molecules.
15:39
And here is the case.
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Using the human PD-1 as target, we immunise NeoMab mice with the extracellular recombined protein of human PD-1.
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We immunise the NeoMab animal and it's background strain BALB/c in parallel.
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And as you can see, after three round of immunisation, the serum titer can reach about 100,000 to 1,000,000 and also highly similar between two mouse tabs.
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And consistent with the ELISA results, the serum can also bind to the PD1 molecule expressed in on the surface of cell lines, which is in its natural confirmation.
16:41
We use the spleen spleenocytes from one of the animals to carry out the antibody discovery by sequencing.
16:55
Analysing of these clones with blocking activity, we found they used a diverse set of human VH and VL genes to encode the antibodies.
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About 7 different combinations observed in this mouse.
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We measure the affinity of antibodies with blocking activity and as you can see, the affinity ranges from 0.1 to 10 more nano more compared to Keytruda and also similar to the antibodies approved for treatment as reported in literature.
17:43
To further test the antibodies, we selected the three clones to produce and tested the efficacy in PD-1 humanised the B6 mouse carry MC38 tumour and overall all of the clones show significant tumour inhibition as compared to the control and the TGI is similar between these clones and Keytruda.
18:17
And for one of the clones 9K6D11 can inhibit tumour at a stronger level as compared to Keytruda.
18:32
In our quest to advanced antibody therapeutics, we conduct another round of selection using another assay to screen for the antibodies with agonistic activity.
18:47
And yes, we can observe obtain some clomes with significant agonistic activity as compared to the BMK.
19:07
And here's the attempts we have tried to break the immune tolerance.
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The immune tolerance of B cells is the mechanism that prevent body to produce antibodies against the self antigen, thereby reducing the risk of autoimmune disease.
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And according the basic immunology knowledge, over 70% of the B cells are removed during the development stage in bone marrow and this is the central immune tolerance.
19:45
However, it brings challenge to antibody discovery especially with targets share that that are highly similar between human and mouse.
20:01
We are.
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Trying different approaches to break the immune tolerance.
20:09
In this case we selected the mouse LAG3 as an antigen.
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This is 40 mouse targets, which means 100% of pulmonology between human and mouse.
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And for the first strategy, we knock out the LAG3 gene from the mouse and it can respond very well after we destroy the target gene.
20:39
And as you can see, the BALB/c have no response to the humanization demonstrate the existence of the B cell tolerance.
20:50
But there are many targets that we cannot knock them out.
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For example, the high similar the highly conserved targets like G, GPC3, they are very important for the development.
21:08
So once these genes are destroyed, the animal will die in early developmental stage.
21:18
And there are other kind of targets expressed on the immune cells like CD20, CD19, which is very important for the immune cell function and development.
21:31
But as you know, there are many targets expressed on immune cells, so we cannot knockout them out.
21:40
In this case, we find another strategy to break the immune tolerance by inducing or modulating the immune system with modulators like antibodies, small molecules and other kind of regions to break the central and peripheral immune tolerance.
22:06
We call that hyper immune.
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We with the hyper immune treatment immune competent animals like BALB/c can respond to the mouse derived antigen like this one.
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And we believe that this approach will significantly broaden the diversity and we are excited about the possibilities the NeoMab platform can offer to this field.
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Our mission is to support our clients in developing therapeutics more efficiently and cost effectively.
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We can provide flexible services and can be tailored to meet the requirement and to study design.
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The NeoMab platform is designed to accelerate antibody development process.
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It offers 40 antibody right from the immunisation of NeoMab mice, reducing the time and reducing the time and resources needed for the antibody discovery.
23:27
And with the 40 human antibody library produced by a NeoMab immunisation, we can skip the humanization step to save a lot of time and money and also reduce the risks.
23:44
Moreover, the timeline is critical factor in drug development.
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OK.
23:52
Our platform is built to support a streamline and integrated approach incorporating resources from GenePharmatech and beyond.
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For example, the target knockout animal from KOAP resources can be used for early-stage target validation, MOA study and also biomarker study.
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And with our vast collection of human targeted humanised resources ready to support the pre-clinical studies if they there is no in vivo study system ready for use, we can establish that system along with animal and test the system in parallel with the antibody discovery process.
24:49
The platform provides a comprehensive support including licence of the animal and provide anybody drug development services and also, we can licence out the products we screened during the validation of the system.
25:17
OK, here is a brief summary of the NeoMab platform.
25:26
Sorry, time is up and welcome to visit us at booth 45.
25:36
Thank you very much.
