Fingolimod blocking Alemtuzumab action what does it tell us about MS?

Willis M, Pearson O, Illes Z, Sejbaek T, Nielsen C, Duddy M, Petheram K, van Munster C, Killestein J, Malmeström C, Tallantyre E, Robertson N. An observational study of alemtuzumab following fingolimod for multiple sclerosis. Neurol Neuroimmunol Neuroinflamm. 2017 Jan 10;4(2):e320.

OBJECTIVE:To describe a series of patients with relapsing multiple sclerosis (MS) who experienced significant and unexpected disease activity within the first 12 months after switching from fingolimod to alemtuzumab.
METHODS:Patients with relapsing MS treated sequentially with fingolimod then alemtuzumab who experienced significant subsequent disease activity were identified by personal communication with 6 different European neuroscience centers.
RESULTS:Nine patients were identified. Median disease duration to alemtuzumab treatment was 94 (39-215) months and follow-up from time of first alemtuzumab cycle 20 (14-21) months. Following first alemtuzumab infusion cycle, 8 patients were identified by at least 1 clinical relapse and radiologic disease activity and 1 by significant radiologic disease activity alone.
CONCLUSIONS:We acknowledge the potential for ascertainment bias; however, these cases may illustrate an important cause of reduced efficacy of alemtuzumab in a vulnerable group of patients with MS most in need of disease control. We suggest that significant and unexpected subsequent disease activity after alemtuzumab induction results from prolonged sequestration of autoreactive lymphocytes following fingolimod withdrawal, allowing these cells to be concealed from the usual biological effect of alemtuzumab. Subsequent lymphocyte egress then provokes disease reactivation. Further animal studies and clinical trials are required to confirm these phenomena and in the meantime careful consideration should be given to mode of action of individual therapies and sequential treatment effects in MS when designing personalized treatment regimens.
If the observation is correct and not a statistical artefact, it must be telling us something about MS.
We all know that alemtzumab is a pretty effective drug at blocking relapsing MS. Likewise fingolimod is pretty effective at blocking MS. However, when alemtuzmab was given following fingolimod, then it looks like alemtuzumab is not working. Why?

Alemtuzumab was started before fingolimod was washed-out and so the disease forming cells are trapped in the lymph glands. Therefore they can't be destroyed by the alemtuzumab, so once fingolimod wears off, cells go back into blood and off MS goes.

First what does fingolimod do?
We have been through this before

Fingolimod is a sphingosine-1-phosphate one (S1P1) receptor modulator and when it binds to its target the receptor is down regulated. As S1P1 is involved in lymphocytes exiting the lymph glands require both S1P1 and a a chemokine receptor. It traps certain types of white blood cell in the lymph glands so they can't get into the blood and they can't get into the brain and so stop disease.

Adaptive immunity depends on regular circulation of lymphocytes between blood and lymphoid tissue in the search for antigens.
When an activating antigen is encountered in the lymph nodes, T cells are retained in the lymph node where naïve T cells become activated and central memory T cells (TCM) are reactivated. Following activation, these T cells return to the blood circulation, allowing them to reach sites of inflammation. 

These cells express CD62L which helps them to traffic into lymph glands via specialised blood vessels called high endothelial cell venules.

Fingolimod does not affect all cells but blocks exit of naive and central memory cells but not effector memory cells. The memory T cell pool functions as a dynamic repository of antigen-experienced T lymphocytes that accumulate over the lifetime of the individual. 

Recent studies indicate that memory T lymphocytes contain distinct populations of central memory (TCM) and effector memory (TEM) cells characterized by distinct homing capacity and effector function. 

This would imply that the disease causing cell is not in the TEM population as fingolimod does not block their movement of cells but blocks relapse. 

The percentages of naïve T cells and TCM in peripheral blood were significantly reduced in patients treated with fingolimod compared with untreated patients, and consequently the percentages of TEM in peripheral blood increased significantly in fingolimod-treated patients compared with untreated patients. While fingolimod reduced the numbers of both CD4+ and CD8+ T cells, the effect was more pronounced for the CD4+ T-cell subset.

We all know that naive T cells would not be the cause of autoimmunity, as they havent been sensitized to a target yet, so that would leave the TCM. Howver we know that CD62L are not the cells entering the CNS.

Fingolimod also blocks B cells entering the blood. These can come from lymph glands but also come from the spleen and fingolimod traps T and B cells in the bone marrow, where numbers go up.

Maeda Y, Seki N, Sato N, Sugahara K, Chiba K. Sphingosine 1-phosphate receptor type 1 regulates egress of mature T cells from mouse bone marrow. Int Immunol. 2010; 22(6):515-25.

Although fingolimod is taken every day, it is eliminated from the system quite slowly, notably because it accumulates in the fat in the brain and elsewhere. 

Once you stop fingolimod, relapses can occur within 1-4 months so it may take a few weeks to a couple of months before cells exit the lymph glands.

How does alemtuzumab work or not work

Alemtuzumab is a lymphocyte depleting antibody and destroys T and B cells. However , you may not know is how it kills white blood cells.

Antibodies can kill by complement fixation. This means that a cascade of small complement proteins are made and these serve to form a membrane attack complex that punches holes in cells. They cause the damaged cells to then liberate their contents and die.

Another way is antibody-dependent cellular cytoxicity

In this case the antibody binds to its target. The antibody is bound by a phagocytic cell by Fc receptors binding to the end of the antibody called the Fc region. The phagocytic cell then attacks and kills the target.

So if we look in a mouse with human CD52 injected with alemtuzumab

Investigation of the mechanism of action of alemtuzumab in a human CD52 transgenic mouse model. Hu Y, Turner MJ, Shields J, Gale MS, Hutto E, Roberts BL, Siders WM, Kaplan JM. Immunology. 2009;128(2):260-70.

If you remove complement (with cobra venon toxin) it has no impact on killing of T (CD4 or CD8) or B cells (CD19) in the blood or the spleen. However if you deplete neutrophils (phagocytic white cell) or natural killer cells which are the ADCC killing mechanism then you don't kill cells.

This is important because whilst you have loads of neutrophils and natural killer cells in the blood you have many fewer in the lmyphoid tissues such as lymph glands and bone marrow.

So whilst alemtuzumab may be good at depleting cells in the blood it will be less effective at killing cells in the lymph glands and bone marrow. Indeed this can be seen above. So whilst you can see that alemtuzumab kills about 90% of CD4 cells in the blood but only 75% in the spleen.

If you look at the depletion in different tissues, it is not too bad in the lymph glands but much less effective in the spleen and bone marrow

So whilst alemtuzumab can clear the blood it may be less good at clearing lymph glands and definately not too good at clearing the bone marrow.

In humans, alemtuzumab is largely gone from circulation within about 2 weeks after infusion.

So if the disease causing cells are sequested in lymphoid tissue for more time than this, then they won't get depleted and then once the fingolimod wears off they enter the circulation again.

Whilst a lot of emphasis is placed on fingolimod acting in lymph glands, alemtuzumab is not that bad at depleting T cells in lymph glands.

However in lymph glands, we and others have found that B cells are less sensitive to depletion than T cells.

Therefore there is relative saving of B cells in the lymph node. In the picture below you can see the cortex and follicles (B cell area black arrow). However the paracortex (T cell area blue arrow) is abit less dense (on right alemtuzumab treated) with low dose alemtuzumab in mice. So the T cells are preferentially being cleared.

Hu et al. 2009

We showed that B cells are depleted even less than T cells notably in the lymphoid tissue compared to blood.

Depletion of CD52-positive cells inhibits the development of central nervous system autoimmune disease, but deletes an immune-tolerance promoting CD8 T-cell population. Implications for secondary autoimmunity of alemtuzumab in multiple sclerosis. von Kutzleben S, Pryce G, Giovannoni G, Baker D.
Immunology. 2016 Dec 7. doi: 10.1111/imm.12696. [Epub ahead of print]

This was shown by others too

Immune status following alemtuzumab treatment in human CD52 transgenic mice. Turner MJ, Lamorte MJ, Chretien N, Havari E, Roberts BL, Kaplan JM, Siders WM. J Neuroimmunol. 2013 Aug 15;261(1-2):29-36.

So B cells are depleted less than T cells and they recover faster, probably because alemtuzumab does not deplete in the bone marrow or lymphoid tissues very well.

Did you know?

Rituximab and ocreliziumab depletes B cells, so has this effect been seen after fingolimod treatment?  It has not been reported yet

Ocrelizumab depletes via ADCC and so one wonders if it's action could be blocked by fingolimod similar to alemtuzumab. So be warned.

But as it is not approved there has not enough time to use it as a switching antibody. But an interesting experiment.

Rituximab depletes via complement and may be better at purging bone marrow cells. 

However as B cell depletion is maintained by 6 monthly dosing so if MS is B cell-mediated, disease won't return, if it is T cell-mediated is could...No reports of this yet. 

So does it point the finger at B cells as the important target in MS?
Is this telling us that MS is a T cell disease or is it all due to B cells?

What does MrT think?

If we did the experiment in the beasties it would be T cells, but in MS?


However, if you are taking fingolimod and need to switch, you need to talk this through before quickly switching to a depletion treatment antibody.

DrK will say even more reason to get a chemical depleter like cladribine back on the table as a small molecule is going to get into those Nocks and Crannies that antibodies like alemtuzumab won't...tick tock, tick, tock.

This rebound will not occur in every body. In this study
there were 36 six people, who had used fingolimod before alemtuzumab and the disease activity was seen in 9 people i.e. 25%. Remember, alemtuzumab is not infallible and relapse (4% in the cardiff cohort) in the first year occurs.

Of these people 8/9 were disease free after the second set of infusions

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