Hi,

Maybe the idea should be reconsidered. To be honest now in vitro models of immunogenicity are not predictive enough for in vivo findings.Of course my advice will be not helpful if in case your specific protein its possible. In case for example more complex proteins like mAb's now its impossible because interaction with immune system is to complicated.

Even if you are using relevant animal model its practically impossible translate immunogenicity findings into humans. One reason is that therapeutic proteins are sometimes human proteins so immuneresponse cover antihuman antibodies release. Second reason cover many differences between animal and human immune response physiology.

From my point of view modeling immune system response based on PBMC will not be predictive enough. Better idea will be comparison of immune response of your protein with any other protein in the market with known profile of immunogenicity and close mechanism and mode of action with the relevant animal model. Then you can check how immune response profiles are different between both proteins. Of course still you cant translate the findings into humans but you can use immune system for your study not only some cells which cannot be a model of systemic response (especially after multiple dosing, see below).

Finally please remember sometimes best way to observe differences in immunogenicity with meaningful impact on clinical response is observation of pharmacokinetics profile. Especially when neutralizing anti drug antibodies (nAB's) have impact on total body clearance.

..... and last advice related to translation immunogenicity from models to humans. Sometimes protein could by very immunogenic and generate immune response for example measured by nAb's .... but after few doses of the protein, level of nAb's decreasing (check ustekinumab case). For example because huge suppression of immune system related to mechanism of action of the drug or as effect of interaction with FcGRIIb receptor. ....

Hope maybe some thoughts will be inspiring to you, :-)

Best regards

Tomasz

Dear Ema A,

Besides your designed tests, I suggest to assess the immunostimulating potential of your protein of interest by some kind of assays like the in vitro proliferation assay using flow cytometry analysis or BrDU test that can detect a slight increase in proliferating T cells.

Hope it would be of help.

Good Luck!

Dear Dr. Tomasz Grabowski, thanks so much for your insights. It is really helpful.

Dear Dr. Mohammad Hossein Yazdi, thanks a lot for your input. I am testing the proliferation of PBMC in response to my protein. it is difficult to see any response. I will try to use a large volume of my VLP-based therapeutic protein

Hi Emy

Your question cover very important field related to biomarkers, because your cytokine role in the study is a biomarker. Before all works you should be sure that your cytokine is really representative for "protective anti-disease immunity". This means that, for example, increasing levels of this cytokine mean that asymptomatic people do not show symptoms of the disease etc (remember about relapses if exist ....). This dependence must be based on the physiological role of this cytokine, which can certainly be applied to other types of diseases. You must definitely justify it in your work.

You will be able to say that, for example, the level of cytokine x ng/ml in the blood corresponds to maintaining a physiological state (asymptomatic cases) if you correlate this with, for example, factors that show how inflammation rising, (CRP or another related to disease symptoms) If for example effect of disease is low level of platelets, try make a relationship between. Finally you can make different pharmacodynamic relationships between your cytokine and pro-inflammatory factors levels and try make a model. If you have small number of data, patients or different sampling points etc try validate such model using population approach.

Maybe some people from Pharmacy department help you, they probably using such methods or know people with experience in population analysis. If you not complete your sampling from patients, subjects try optimize sampling plan for cytokine and covariates with specialist in population approach. Population methods gives you more insights and observations for discussion, if implemented.

https://www.kuhp.kyoto-u.ac.jp/~yakuzai/english/staff.html

Hope its partially answer to your question ....

Best regards

Tomasz

Hi,

So you have .. please correct me if Im wrong:

A - asymptomatic patients [afebrile] PCR positive

B - asymptomatic patients [afebrile] PCR negative

Of course you can try make comparision between AB groups in relation to cytokine levels but please remember cytokine release is continous dynamic proces so findings from your comparision will depend on sampling method (continuous - many samples in different time points, or static, more categorical in few or single specific timepoint).

Best regards

Tomasz

Hi

Now its clear, sounds good!

Tomasz

Hi Atira Ety

given that the more persistence of antigen , the more effective response will emerge, to investigate memory cells, evaluate CD3 and CD28 as T cell maker and also CD27 and IgD as B cell maker.

I hope it will be useful