Slide 1

Dear Chairman, Ladies and Gentlemen, thank you very much for the kind invitation to present to you some of our results regarding the immunoliposome targeting to mesangial cells. It’s a new strategy to be useful for directed drug delivery in the kidney.

Slide 2

First we have to ask ourselves the question why may it be useful to have a tool for directed drug delivery?

Slide 3

In my lab I have had a long standing interest in the pathogenetic role of matrix metalloproteases in glomerulonephritis. We treated anti-Thy1.1 nephritic mice with MMP inhibitors and showed the course of the disease was markedly attenuated by these compounds with a decrease in proliferation and induction of apoptosis of mesangial cells. However, MMP inhibitors are cumbersome to administer, they need to be given intraperitoneally. We don’t have any good compounds that can be given by the oral route, they have side effects and have never took on in humans apart from a few oncology trials phase I, phase II.

Slide 4

So in humans we couldn’t use these MMP inhibitors, so we did a triple cross-over study with 10 patients with glomerulonephritis and showed that ACE inhibitors were able to reduce MMP activity considerably. This was not the case when we applied an angiotensive receptor blocker. However, ACE inhibitors are not used to inhibit MMP activity, they’re used for other things and if we use that individual MMPs here control, here membranous and here membranoproliferative glomerulonephritis, some MMPs were upregulated in GN and some of the MMPs were downregulated. So in other words we don’t have any good compounds that can selectively modify MMP activity and we have no good compounds in humans. That’s why the aim of our study was to design a vehicle for a future, selective and specific MMP inhibitor in GN.

Slide 5

For this reason we resorted to the liposome technology.

Slide 6

Now, what’s a liposome? A liposome is simply a microparticle formed by a phospholipids membrane bilayer. Now, an immunoliposome is such a liposome coupled to an antibody or to an Fab fragment via a spacer for instance polyethylene glycol. So a liposome connected to an antibody and then you have an immunoliposome. There’s quite considerable literature in oncology whereby tumours can be selectively targeted by the use of such immunoliposomes directed against tumour antigens but there wasn’t anything in the literature with regards to mesangial cell derived kidney disease.

Slide 7

As already heard by Tammo Ostendorf in the previous talk, the rat mesangial cells are characterised by the expression of the Thy1.1 antigen in the glomeruli of rats. Now, our hypothesis was that these rat mesangial cells can be targeted by the use of the Thy1.1 antigen. By coupling of an anti-Thy1.1 antibody in fact, we used the Fab fragments of an OX7 IgG to a liposome we can direct molecules to the glomeruli.

Slide 8

So we constructed OX7 immunoliposomes by the attachment of Fab fragments of this IgG to a liposome. First we analysed the binding of liposomes and immunoliposomes in cultured mesangial cells. Here a Thy1.1 negative cell and there was no binding of liposomes or immunoliposomes. However, when we used Thy1.1 positive rat mesangial cells as measured by fluorimetry of cell lysates, we had quite a marked binding of OX7 immunoliposomes and just a little binding of empty liposomes. These results were subsequently confirmed by FACS analysis showing a marked binding of OX7-IL to the rat mesangial cells but not to the Thy1.1 negative cells and there was not much binding of immunoliposomes.

Slide 9

Subsequently, we analysed the kinetics of the uptake of these constructs. Here over time, over 7 hours as analysed by fluorescence of cell lysates and you see there is a small uptake of liposomes however, a much steeper uptake of OX7-IL. So these cells, these immunoliposomes bind to Thy1.1 positive cells and get internalised. We confirmed the internalisation of immunoliposomes by confocal microscopy. Here the uptake by 37°. You can see there’s a much higher uptake of immunoliposomes in the cytoplasm of cultured mesangial cells than just the uptake of liposomes.

Slide 10

So, these constructs actually bind to the mesangial cells and get internalised. Subsequently, we went on to in vivo studies and studied the pharmacokinetics of radioactively labelled liposomes and immunoliposomes. Here time 0–100 minutes and here the radioactivity in the blood. You see that liposomes are pretty stable and have a quite a long half life in the circulation. However, OX7 immunoliposomes in blue rapidly disappear from the circulation.

Slide 11

When you co-inject OX7 immunoliposomes, there’s a 10-fold excess of OX7 antibodies, the half life goes up and is in the order of just liposomes. If we co-inject OX7 immunoliposomes as non-specific antibodies, the kinetics is not much influenced.

Slide 12

So we determined several pharmacokinetic parameters here half life long in liposomes, short immunoliposomes and the immunoliposomes plus an excess of Fab OX7 about the same half life. This is mirrored by the blood clearance very fast liposomes, immunoliposomes plus OX7 for competitive binding and pretty low or compared to liposomes low, if you just look at the immunoliposomes. The systemic volume of distribution is similar.

Slide 13

Now, the next question we have to ask is actually do these immunoliposomes go into the glomeruli? Here immunofluorescence of healthy Wistar rats 2 hours after injection of these constructs. When we inject PBS, there’s only background fluorescence from tubuli, the FITC-liposomes there’s not much change to PBS. However, when we inject FITC-Immunoliposomes, you see a lightening of the green fluorescence of all glomeruli. If you co-inject immunoliposomes Fab against OX7, this is again abrogated.

Slide 14

So actually these immunoliposomes go into the glomeruli and we can see in a more or less mesangial fashion. – localisation by confocal microscopy again in healthy rats. Here you can see the staining of the mesangium using a Texas red labelled OX7 antibody and here the staining for our immunoliposomes FITC labelled and you can see this corresponds quite well and we have a good merge when you merge red and green which means – immunoliposomes get deposited in a mesangial fashion.

Slide 15

When we enlarge this picture, we can even see at the cellular level in the mesangial cell in green these immunoliposomes actually in the cytoplasm. When we add a Hoechst nuclear stain, we can see not only cells are positive, there are some negative cells as expected, the endothelial cells and the epithelial cells. So also in vivo these immunoliposomes go actually into the mesangial cells. Subsequently, we investigated the biodistribution of radioactive compounds to see if they go also into other organs. Here the quantity of microparticles per gram of organ and here per total organ. You see several columns in light blue liposomes, in dark blue OX7-IL, in green co-injection with non-specific antibody and here OX7 excess with OX7-IL. You can see there are 3 organs with a very high biodistribution of immunoliposomes, these are spleen, lung and liver, --- analysed per gram of organ or per total organ, spleen, lung liver. There was also deposition in the kidney but very little as expected because the mesangial cells are only a few percentage of the kidney tissue.

Slide 16

So as far as the biodistribution is concerned most get deposited in spleen, lung and liver.

Slide 17

Now if we analyse the tissue distribution how much we have in a given tissue, then we can see here in the renal cortex as shown before nothing of liposomes but a very high density of positivity in the glomeruli. There’s not much positivity in the liver, there are some – cells which are positive now their position at least visible in the lung and there was as expected a marked deposition of liposomes and also the immunoliposomes in the spleen. There was no OX7-IL deposition in brain, heart, thymus, we unfortunately didn’t analyse the bone marrow.

Slide 18

So if we look at the tissue distribution per tissue, we only have a high concentration of these immunoliposomes in the glomeruli and in the spleen. Now at the end, we also examined the Thy1.1 tissue distribution and we had a very high expression of the Thy1.1 in the brain and in the thymus and we had some in the spleen, lung, a little bit in the liver, heart, kidney but not in the heart. Here the brain quite positive for the Thy1.1 antigen heart negative, the glomeruli in the kidney positive.

Slide 19

There was slight positivity in the liver, some -- cells, the spleen was positive and the thymus was markedly positive but there we had no OX7-IL deposition.

Slide 20

So to summarise so far we have a very strong Thy1.1 expression in the thymus and brain but no immunoliposome deposition because these organs are not accessible to the OX7-IL. We have a moderate Thy1.1 expression in kidneys, maximal in glomeruli where we also have immunoliposomes in the lungs, very little tissue distribution, tissue have concentration and moderate Thy1.1. in the spleen where we also have liposomes, weak expression in the liver and heart of the Thy1.1 antigen.

Slide 21

So if we summarise the pharmacokinetics we have the specific targeting that is responsible for the fast OX7-IL clearance. These immunoliposomes get cleared by binding to the Thy1.1 antigen. In the biodistribution OX7 is mainly found in spleen liver lungs and kidneys. However, if you look at the level of the tissue, we only have a very high tissue density in the glomeruli and in the spleen. So, the next question was can we achieve a pharmacological effect using these constructs?

Slide 22

We loaded doxorubicin into OX7-IL and in liposomes we used doxorubicin because it’s easy to be loaded in these constructs because of technical reasons it precipitates and we can achieve a high concentration of this drug in these constructs and we can use a negative pharmacological effect damage in case it works. If we look at the PBS injection empty liposomes, empty immunoliposomes of course no damage, if we look at the injection of free doxorubicin there was no damage in the kidney, liposomal doxorubicin no damage but if we load doxorubicin in immunoliposomes there was quite a marked damage. We used quite a low concentration of doxorubicin 250 μg/kg body weight. There’s an animal model where people use 30 times higher dose and after a week they only observed slight changes in these podocytes.

Slide 23

We had marked changes within 24 hours using a 30- fold less dose. Here enlarged in the kidneys we have very strong glomerular toxicity, quite a marked destruction of all the glomeruli by the use of doxorubicin in the immunoliposomes and not by the other forms.

Slide 24

We quantified this damage, here cells per glomeruli and the animals that received doxorubicin in immunoliposomes had only about half the cellular content in the glomeruli. Here the percentage of glomerular damaged area was only notable in the animals that received doxorubicin in the immunoliposomes.

Slide 25

So we were able to achieve a clear pharmacological effect by the incorporation of doxorubicin into immunoliposomes. We checked the other organs at the histological level and we found no toxicity in the liver by any of these constructs also no toxicity in the lung, no toxicity in the spleen as far as it was visible at a histological level.

Slide 26

So to summarise in vitro we have a good specific binding and uptake of OX7-IL with regards to Thy1.1 positive cells. In vivo in rat mesangial cells we have a strong affinity and specificity of OX7-IL to glomerular mesangial cells. We have an accumulation of immunoliposomes in the cytoplasm of mesangial cells and we have a distinct pharmacological effect as expressed by an extensive glomerular damage when we use doxorubicin loaded OX7-IL. Apart from the glomeruli there was only marked deposition of immunoliposomes in the spleen but if you look at the tissue density not in the other organs.

Slide 27

Where do we go from now? Now we want to include other molecules into these liposomes and immunoliposomes. Here we can see fluorescence under UV light of empty liposomes here with a hydrophilic compound carboxy-fluorescin in immunoliposomes and liposomes and here a small lipophilic molecule diphenyl-hexatriene also successfully loaded in liposomes and immunoliposomes.

Slide 28

So now we are able to load other small molecules into these compounds. We checked in some preliminary results the carboxy-fluorescin containing immunoliposomes and they quite nicely bind also to the mesangial cells and get taken up as you can see here.

Slide 29

Also in the kidney in healthy rats we have also mesangial deposition of fluorescin loaded immunoliposomes.

Slide 30

So I would like to conclude my findings. The Immunoliposomes represent a highly promising carrier system for the delivery of therapeutic agents to glomerular mesangial cells and we think the mesangium is quite a good target for immunoliposome-mediated drug delivery.

Slide 31

Slide 32

Chairman: Thanks very much indeed. Peter so we have questions. There are two microphones down the middle of the aisle. Please give your name and where you live. I thought that lady was going but she’s not. Can I ask? I didn’t quite understand, you said the biodistribution of the liposomes was actually in the lungs, livers, spleen but when you looked at the tissue sections it was not there it was only in the kidney. What happens to them? Do they get broken down or disappear?

Dr Marti: I mean basically what we want with the immunoliposome is we want to have a very strong density within a target cell. We reached this only in the mesangial cells as we wanted we had a lot of immunoliposomes within a cell. Also these splenic cells the reticular-endothelial system they also capture a lot of these immunoliposomes so only in these two organs at a cellular level we had a very high density. If you look at the big organs like liver or lung, overall they capture quite a lot for the high part of these immunoliposomes but at a cellular level this wasn’t very dense. So they didn’t get many immunoliposomes per cell but because these organs are quite huge, if you add it up they capture lots of immunoliposomes but at a cellular level it wasn’t very dense.

Question: Do they pass through the blood-brain barrier?

Dr Marti: No.

Chairman: No. Sorry a question there.

Question: Sorry, Alison Seven from Dundee in Scotland. Is there any evidence that when the mesangial cells internalise the liposomes they become activated at all?

Dr Marti: What happens when they internalise it?

Question: Yes do they produce cytokines or anything as a result?

Dr Marti: I mean it’s open for speculation it can be taken up by endosomal, by the endosomes and then they might join to lisosomes and we don’t know if some therapeutic agents are liberated then or metabolised and destroyed, this we don’t know yet but at least in the case of doxorubicin it must have some nuclear action so actually in this case it got taken up and the drug must have been liberated and taken to the cell nucleus. So, it needs to be shown in the middle for each medication.

Chairman: Thank you. Other questions and comments? Can I ask if you’re thinking of applying this to the human disease what would be a good target in the human mesangial cell?

Dr Marti: I don’t think we need an antigen that is exclusively expressed on human mesangial cells but we just need an antigen that is expressed on a high amount on the human mesangial cells. Up to now we haven’t defined yet such an antigen because we have just fenestrated the endothelial these constructs have access to the mesangium and then if they find an antigen that is expressed to a significant part they’ll bind to the mesangial cell.

Chairman: So Thy1.1 is only in rats?

Dr Marti: In rats yes. We have to find a way to saturate the spleen for instance, before we inject the immunoliposomes so they actually go there and not too much into the spleen.

Chairman: Any other questions? Ok. If there are no more questions thank again very much indeed.