Alemtuzumab:The Irony of Humanization Part III

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So we have got as far as saying that people who recieve alemtuzumab can make an anti-drug response (Part I and Part II) and we will explain this (part V) and show the importance of these (Part V, VI). Part III and IV are lab lessons, so if you don’t want to read the science you can have a rest

But you have asked….what have we been doing?

Alemtuzumab is very effective for most people and about 50% of people do not need more treatments for many years, showing it can effectively block relapsing MS. Have another doses and that is 75-80% of people effectively treated for a number of years.

However, we reported that about 60% of people make a blocking response within the first month of the first infusion and about 80-85% of people make an anti-drug response within one month of the second infusion. Remember 50% of people fail treatment and need another infusion. After the first infusion it has been shown that anti-drug antibodies disappear by 11 months after infusion. But after the second infusion at 11 months about 30-75% of people still have anti-drug antibodies (ADA). This means they may stop the alemtuzumab working in some people.

So we had to try and work out if we could measure these before people got the next infusion. We could perhaps predict if the alemtuzumab infusion was going to work. So how would we do this?

NDG and I approached our Antibody Wizz DrAngry, he’s very nice really, to see how best to do this. We could try and make an anti-drug antibody and this would take months to do and whilst there were some reagents they cost a fair bit and for some antibodies these reagents are not readily available.

The manufacturer was in denial and still is (ECTIRMS 2018 below) that these ADA were important to some people

This image has an empty alt attribute; its file name is poster-1024x143.png

We got no support to do this, so we used “student power”. I must say a big thanks to Barts and QMUL students who made these reagents as part of their lab projects for their degrees. However, we are not just slave drivers…and we did send Gauri (Saxena) to Italy to present her work to the Neuros at the Charcot Foundation. That’s why our students love us and importantly they have a paper to show for their efforts.

Now before telling you about the technology, you have to go to School to learn about the structure of an antibody molecule.

The antibody molecule is made up of two chains a big one called the “heavy” chain and a small one called the “light” chain. There are regions called constant regions that mediate the effector function of antibodies, like killing the cellular target. Then there is the variable region, which contains the antigen-binding site.

Notably, there are three regions on each arm of the heavy and light chains called the complementarity determining regions. There are CDR1, CDR2 and CDR3, which are the bits kept in a humanised antibody These are the bits that bind the target. So the CDR regions in alemtuzumab come for the original rat antibody and the rest is from human antibody proteins

When antibodies are commercialised the amino acid sequence is revealed in the patents or the papers describing the antibody. So we found these and synthesised the DNA of the antigen binding bits and on to each chain we added a glow in the dark molecule. This molecule which is called luciferase was originally found in little devils …OK…in the firefly. The luciferase acts on luciferin to create light

Actually the version we used was made in a deep sea shrimp…It vomits the luciferase-like molecule (enzyme) and another molecule (substrate) that is activated by the luciferase and emits light, blinding the prey and giving the shrimp a meal. The shrimp variant is smaller (nanoluciferase), more stable and brighter than the firefly variant.

A vomiting shrimp…Did you know that MD2 and I went to a meeting in San Franscisco and we saw a picture of a vomiting shrew just before Lunch?. I’m scarred for life:-)

So we have the heavy and light chain of the antibody in this case alemtuzumab containing two nanoluciferase molecules linked together to form what we call a Globody (Gosh…you would think I work on Maddison Avenue as it’s like Madmen Advertising Ideas..Yep they came up with Irony of humanisation too..Genius). This acts as a bait for the anti-drug antibody, so the alemtuzumab globody is bound by anti-alemtuzumab antibody and the ustenikumab globody (specific for interleukin 12/23) is bound by anti-ustenikumab antibody and not the anti-alemtuzumab.

Mix the globody and blood serum and the anti-drug antibodies are now labelled. This mix is added to plastic plates coated with protein G, which is an immunoglobulin binding molecule from Streptococcal bacteria, so the ADA and other antibodies are stuck to plastic. Wash the plate and add the luciferase substrate and bingo you get light. You now have a tool to quantitate the amount of anti-drug antibody.

50 microgrammes of anti-alemtuzumab, which is about 30 times the theorectical maximum amount of alemtuzumab that can every be produced from infusion gives about 20,000 light units. The highest we have measured in a person with MS is about 10,000,000 light units. Is this important? You have been told no…numerous times by the inventors and manufacturer. Are we so sure?…..We shall see soon, but you need a bit more information before we start on that.

The beauty of this system is that you can use the same assay whether it is mouse, rat, dog monkey or human being tested and importantly we can rapidly make the globody to any therapeutic protein, whether it is a rodent, humanised or human antibody. We can do this as fast as you can say “student power”….or maybe give us support to do more and do it more quickly?

Give us a bit of support and we can make a device that will measure the ADA at the point of service, blood prick, test, and get your infusion in the knowledge that it is likely to work. You laugh…but I have seen the kitchen table top version prototype. It is nice to work with some Bright Guys.

Importantly we have made a tool to help our neuros at BartsMS or elsewhere. This is what we do. Basic science to support our clinical science. They give us the problem, we try and get a solution.

Alemtuzumab comes in at about £21,000-£28,000 a year in UK, at least until the UK-US trade deal:-(In the US the cost is nearer $90,000 or more. Would you rather have a test to see if you are going to respond or take the risk….fail the treatment and end up with unneccessary disease activity and a very big hole in your pocket?

However, just think that some antibodies cost about £350,000. Wouldn’t you want to know if it is not working? No wonder, we have had a job finding an example…However that’s another story…I think I’ll call it DrAngry…Dead in a Ditch 🙂

Anyway the paper is open access have a read. The good news is at the time we were doing this, we did not have bloods from people in our care, who we thought would not respond. This is the first part of the methodology, only one more bit to go. However, it says the percieved problem isn’t a big issue

CoI: None relevant, but DrAngry has filed patents on the technology.

Have a read it is open access.

Saxena, G.K., Theocharopoulos, I., Aziz, N.T. et al. GloBody Technology: Detecting Anti-Drug Antibody against VH/VL domains. Sci Rep10, 1860 (2020). https://doi.org/10.1038/s41598-020-58041-3 .

The occurrence of anti-drug antibodies following administration of therapeutic monoclonal antibody to patients is a growing problem that is attracting attention from frontline clinicians. Ideally, an initial indicative point of care test would provide guidance to seek testing approved by the regulatory authorities. Here we describe a platform for the detection of IgG anti-drug antibodies that may provide an initial screen for all therapeutic monoclonal antibodies. Synthetic genes encoding Nanoluciferase polypeptides were inserted between the variable heavy and light domain encoding region of known antibody drugs (alemtuzumab and adalimumab) to generate recombinant single chain GloBodies, which retain the drug antibody paratopes and Nanoluciferase activity. In the presence of anti-drug antibodies, the GloBody is bound by specific IgG in the sample. These complexes are captured on immobilised Protein G and the luciferase activity determined. The amount of light generated being indicative of the anti-drug IgG antibody levels in serum. It should be possible to assemble GloBody reagents for all therapeutic monoclonal antibodies and adapt the capture phase to include additional specific isotypes. The assay has the potential to be developed for use with a drop of blood allowing initial pre-screening in a point of care setting.

DrAngry on the Kangmobile

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12 comments

  • Gosh! It’s a read like this, most of which I’ve comprehended, that makes me feel humbled by the work of yourself, MD2 and all your students – thank you so much!
    No this doesn’t offer a cure for MS, no it’s not applicable to so many with this disease, nor is it an add-on therapy to restore what we’ve lost, but I am thrilled at the thought of this vital refinement to the provision of Alemtuzumab. If I require a third dosage at some future point I’d definitely want to be tested.
    It’s a thumbs up to anything and everything that eases or facilities our lives as PwMS. Yes, a cure is the ultimate goal, but as I’m keen to remind myself MS is the only neurodegenerative disease for which there’s any treatment at all.
    Best wishes for all your ongoing work.

    • In regards writing about a cure we published this before the blog started.

      I would direct you to Pryce et al 2005 J Neuroimmunology where for over twenty years we have been able to cure EAE, if too much progression has not kicked in.

      It suggests the processes that need to be taken and how to do it if you don’t know what is causing the problem.

      This solution has been around since the 1960s.

      However BSE has put a stop to the approach.

      Furthermore it is.difficult.to do this in MS as it is fraught with failure and is best first tested in a much simpler system where a simple target is known.

      We actually have done this and the paper will be submitted as soon as the last person finishes reading it.

      However, I hate to say that it will take a major effort to revive this, especially as people keep messing it up.

      Would you want to invest in a company selling an approach when there is a graveyard of failed attempts that put people off.

      Also you need to manufacture at least two things. If you have a monotherapy it does not work properly.in animals so why would it work in humans, I have told two companies to their face that they were missing a trick and.making abcatastrophic major.mistake. They are no more.

      One of the reagents needed is no longer in production and we could not.convince the companies to revive the product when we tried a few years ago.

      Maybe if a big.swinger asked they would be more obliging. I have said this to another person with grand plans that their approach would fail without the depletion step. There was a deadly hush that I dare speak to a science demi god in such a way. Come back in five years and this dream will be a broken.:-(. It will also fail because they are using.tje wrong antigen

      We tried and idea of myelinating stem cells but they didn’t produce enough protein maybe we should write this up.

    • it is a good drug but it can be used more safely However, this is a proof of principle and we can do the same of other agents.

    • A price has not been set we have been doing this for free, Dr Angry spends his weekends doing it. Commercially, it shouldn’t be any more than a standard immunological test. This would be a tiny tiny fraction of the £30,000 cost of 3 infusions of alemtuzumab. Hey if Genzyme get it together they could offer it as a service you can ship the bloods in the post as antibodies are incredibly stable.

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