Alemtuzumab: The Irony of Humanization Part IV.

A

You asked us what have you lot been up to?

DrAngry in Designer Dickie. He likes a bit of Couture. The next offering (Part V) was drawn
using the Spring 2020 Colours

In Part I we showed that alemtuzumab, which was the World’s first humanised antibody, designed to reduce the immunoogenicicty of the orginal rodent antibody, is probably the World’s most immunogenic therapeutic antibody. In part II we found the suggestion that people who made high levels of neutralizing (inhibitory) antibodies , stopped depleting their lymphocytes. This triggered a company presentation to say that they were not important. However, we didn’t buy into that and decided we should make tools to independently measure the level of anti-drug antibodies. Part III which was published on wednesday showed that we made globodies to measure binding antibodies. However, in the way we did it this would detect binding (that bind to the non active sites of the variable region) and neutralizing (that bind to the antigen binding site that will block function of the alemtuzumab).

This is still a lab lesson and the publishers are abit slow, so if not interested wait until the next installment, however if you are taking alemtuzumab you may be interested.

So the next stage was to make a neutralizing assay, and to do this we needed a cell that made CD52. Easy you say, you can look for cancer cell lines that sceintists can get their hands on. I guess you may have heard of HeLa cells, which was a cancer cell from Henretta Lacks.

HeLA cells

We could get cancers of white blood cells that would express CD52. For example JURKAT is a T cell cancer

BIOGPS NCI60 cancer cell lines

However we wanted an adherent cell line that sticks to plastic, so we made our own. We took CD52 DNA from a cell and put it in to a Chinese Hamster Ovary (CHO) cell line. So we could test where the anti-alemtuzumab drug antibodies could block the binding of our alemtuzumab reagent to CD52.

This paper describes one variant of the assay that we developed, in this study we blocked fluorescent alemtuzumab from binding to CD52.

Do a wash and where there is ADA it blocks binding

We are now ready to measure binding and neutralizing anti-alemtuzumab antibodies. In our intial studies, it was good to know that people who we were giving the alemtuzumab to where still responding to alemtuzumab, but could we find an example where alemtuzumab-specific ADA was stopping the drug from working.

The answer was rather surprsing to me. More in part V.

A cell-based assay for the detection of neutralising antibodies against alemtuzumab. Ali L et al. Biotechniques 2020. BTN-2019-0122.R1

Ali et al. 2020 biotechniques from BartsMSBlog

Abstract:

Background: The humanized anti-CD52 monoclonal antibody alemtuzumab depletes lymphocytes and is currently used to treat relapsing multiple sclerosis (MS). During treatment in MS patients, anti-alemtuzumab antibodies may reduce effective lymphocyte depletion. Here, we describe an adherent cell based assay for the detection of neutralizing antibodies against alemtuzumab.

Results: Alemtuzumab-Alexa 488 conjugate binding to the CHO-CD52 cell surface was inhibited by anti-alemtuzumab antibodies in patient serum.

Conclusions: A stable CHO cell line expressing human CD52 has been established and used to detect the 10 presence of anti-alemtuzumab neutralizing antibodies in the serum of a MS patient treated with 11 alemtuzumab. This platform provides a cost effective quantitative assay for detecting neutralizing antibodies for routine screening of serum from patients treated with alemtuzumab.

Lay abstract: Therapeutic monoclonal antibody are the most advanced targeted therapies available, and they are currently used for the treatment of numerous diseases and conditions including relapsing multiple sclerosis. However, they all have the potential to cause immunogenic reactions and the generation of antibodies, which bind and reduces therapeutic efficacy. As a result, patients do not receive expected benefits from treatments, and the risk is cumulative with repeat dosing. The timely detection of anti-drug-antibodies (ADAs) has the potential to avoid these major risks. Here we describe a cell based method for detecting anti-alemtuzumab neutralizing antibodies

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