Hello everyone,
I am working on hPBMC that I put in culture with PHA for 72h (to increase the PD1 expression) after what I treat 30 - 45 min with Nivo or Pembro before to start my staining for T cells.
I'm looking for an Ab anti-PD1 in flow-cytometry or IHC which could bind PD1 even if the cells are treated before with Nivo or Pembro.
I already tried with the clone Eh12.2H7 from Biolegend and EH12 from BD but when my cells are treated I can't get a signal.
Is someone know and already stained with a commercial Ab who bind on different epitope than therapeutics Ab?
Thank you for your help.
Regards,
Hi Cyril,
Firstly, I cannot answer your question because I do not know. However, I looked up the structure and the binding regions for Nivo/Pembro, both the antibodies bind to different loops but overlap in certain regions. To find an antibody that can still bind exclusively without any overlaps may be difficult. I think your best bet would be to contact several prospective manufacturers for full information on their Abs.
Link for illustration: https://www.ncbi.nlm.nih.gov/corecgi/tileshop/tileshop.fcgi?p=PMC3&id=562599&s=71&r=1&c=1
Do not know if that helps at all. Good luck!
Hi Darellynn,
Thank you very much for your time and your answer.
I already contact several manufacturers and it look exactly as you say, no possibility to bind after Nivo and Pembro. I will continue to look after and will let you know if something new happened.
Kind regards,
Cyril
Hi Cyril,
The companies are correct, Pembro and Nivo have the highest binding affinities compraed to the common PD1 abs (like MIH4), we experienced the same. However, if the goal is to detect PD1+ cells, you could still detect Pembro-bound T-cells, using fluorescent anti-human secondary (I believe Pembro is IgG4) to detect them, which they obviously won't tell you. I've used it successfully. Make sure you include excellent isotype controls to ensure specificity.
Hi Sri Krishna,
Thanks to participate at this topic. Effectively we can use a secondary to stain T cells bound by Pembro or Nivo. But our aim was to now if an common anti-PD1 was able to bind in a different way than this Therapeutic Ab.
Nowaday, I haven't found one.
Hi Cyril Deceneux,
I was wondering, what is the concentration of Nivo that you are using when you are incubating Nivo with your PBMCs? Since up to 1mg/ml Nivo we can still partially detect PD-1 positive T cells with our anti PD-1 antibody (clone Eh12.2H7) while you wrote that Nivo completely blocks your PD-1 signal.
Thank you for your help!
Best,
Kitty
Hi Kitty,
Sorry for the late answer, we used Nivo at 10μg/ml and Pembro at 12.5μg/mL.
Best regards,
Cyril
Dear Kitty,
we're also using 10 µg/ml. It is a commonly used concentration for functional tests.
I believe that the signal that you see, must be an artefact, background, compensation issue or something else.
We also observed that commercially available antibodies (we tested 5 different clones, including the EH12 clone) can't bind in presence of both pembro or nivo.
And like @Darellynn Oo said, it is very likely that it's simply impossible that there is an antibody clone that can still bind PD-1 without any overlaps...
I think the only way to go is the mentioned detection using a secondary antibody.
Kind regards,
Henning
Thank you Henning Zelba and Cyril Deceneux for the usefull answers. We will have another look at our analysis/compensation etc. Also we only used an FMO and not an isotype control, which may explain our 'false positive' signal.
Dear all,
by the time, Cyril Deceneux asked, we had already prepared a manuscript dealing with exactly this problem. It was accepted last week, you can access it here:
Article Accurate quantification of T-cells expressing PD-1 in patien...
KR Henning
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