WO1985005453A1

WO1985005453A1 - New compositions of liposomes having a specific action with regard to the proliferation of given cells

Info

Publication number: WO1985005453A1
Application number: PCT/FR1985/000125
Authority: WO
Inventors: Patrick Machy; Lee Daniel Leserman
Original assignee: Institut National De La Sante Et De La Recherche M; Centre National De La Recherche Scientifique
Priority date: 1984-05-21
Filing date: 1985-05-21
Publication date: 1985-12-05
Also published as: FR2564319A1; EP0181902A1

Abstract

The compositions of liposomes comprise at least one compound which is active with regard to the cell proliferation, and is encapsulated in a liposome to which is bound a ligand capable of specifically targetting the cells to be saved.

Description

"New compositions of liposomes with a specific action against the proliferation of given cells"

The subject of the invention is new compositions of liposomes endowed with a specific action with regard to the proliferation of given cells.

It also relates to the use of these compounds as biological reagents, in particular, to allow the selective growth, in vitro, of one or more cell populations.

The positive selection from a heterogeneous culture, from a given cell population, allowing its survival and proliferation has given rise to numerous studies. Most of the techniques developed to date are based on the recognition of the cells to be selected by a specific ligand, for example, a lectin, a hormone or an antibody, the ligand then being conjugated to a drug, a toxin, a fluorescent compound or various protective compounds.

These techniques make it possible to develop systems which can be used as models, but are not suitable for larger-scale applications. Their major drawbacks reside in particular in the insufficient capacity of transport of the ligand, the difficulties of the chemical coupling of the ligand, the implementation of cell lines or particular equipment.

In previous work, the inventors have shown that it is possible to inhibit the proliferation in vitro of a cell population, using liposomes. These are liposomes having on their surface an antibody capable of specifically targeting a given cell and containing, encapsulated, a compound inhibiting cell proliferation, namely, methotrexate (in abbreviation, hereinafter, Mtx).

The results obtained showed that the proliferation of cells which are recognized by the antibody used was inhibited with high specificity.

The state of progress of the work of the inventors in this field has enabled them to study the possibilities of applying compounds ————— on the contrary promoting cell proliferation which has led them to the development of new compositions of liposomes.

The invention therefore aims to provide new compositions in the form of liposomes capable of selectively ensuring the survival of given cells.

It also aims to provide biological reagents based on these compositions which can be used, because of their high specificity of action and their effectiveness, to ensure in vitro the survival of a given cell population or of a sub-population. even present at very low concentration in a culture.

The compositions of the invention are characterized in that they comprise at least one compound active with respect to cell proliferation, encapsulated in a liposome to which is linked a ligand capable of specifically citing the cells whose survival is desired .

These compositions bind specifically in vitro to the cells to be saved and advantageously penetrate inside these cells by delivering the compound. active capable of ensuring their survival.

The action exerted is sufficiently powerful to save a population of given cells, even in the presence in the culture medium of lethal concentrations of inhibitors.

For the preparation of the compositions of the invention, the active compound is used according to a concentration chosen so as to be able to save given cells in the presence of toxic amounts of inhibitors such as Mtx or a derivative, in the medium.

In a preferred embodiment of the invention, the active compound comprises or is formed from tetrahydrofolate, in particular N ⁵ -formyl-tetrahydrofolate, abbreviated below THF or a derivative thereof, or alternatively a salt of this derivative.

The use of tetrahydrofolate or its derivatives in the form of a salt, in particular a sodium salt or a potassium salt is particularly advantageous since it makes it possible to avoid any problem of aggregation and precipitation of the liposomes. The ligand attached to the surface of the liposome is chosen from compounds capable of specifically recognizing the cells which it is desired to target. Suitable ligands include monoclonal antibodies, lectins, hormones, protein transport and generally any compound having an affinity for a given phenotype on the cell to be saved.

Advantageously, monoclonal antibodies are used corresponding to determinants expressed by the cells whose survival is desired. Only the cells expressing these determinants are saved and proliferate in a manner analogous to that observed with free THF, when they are exposed to amounts of free Mtx which would be sufficient to kill them in the absence of THF. On the contrary, the growth of cells not expressing the targeted determinants is inhibited by Mtx present in the culture medium.

Liposomes are produced in the usual way. Preferably, liposomes are used as prepared according to the article by Machy, Barbetet Leserman in Proc. Nat. Acad. Sci. USA 79 (1982) p 4148-4152.

They are liposomes based on small phospholipids, which promotes the delivery of the active compound. Their dimensions are preferably less than 4000Å, more especially less than 1000 Å, or even 600 Å. These liposomes are advantageously conjugated or coupled by covalent bond to a compound making it possible to ensure the bond with the chosen ligand. Protein A is preferably used especially when the ligand is made up of an antibody capable of fixing this protein.

To identify the cells to which the liposomes are fixed, the latter contain, in addition to the active compound, also encapsulated, a marker.

Suitable markers include fluorescent compounds, a preferred compound of this type being carboxyfluorescein. The concentration of marker is advantageously of the order of ImM to 200 mM.

These liposomes therefore make it possible both to protect and to mark the selected cells. The use of small and high purity liposomes promotes obtaining a high specificity of action.

In this regard, use is advantageously made of a purification of the liposomes on a gel chromatography column. The foregoing arrangements make it possible to obtain liposomes which can be used for the development of highly effective biological reagents for selecting a given cell population while ensuring its survival.

Thanks to these reagents, it is possible to deliver the active compound to the cells of this population without the need to chemically modify it for its administration, which ensures maximum efficiency.

The work carried out has shown that the active compound encapsulated in the liposome is as effective as when it is free.

According to another particularly interesting aspect, the reagents of the invention make it possible to take advantage of the advantages of liposomes, namely, in particular, their relatively easy preparation, their high transport capacity, the means which they offer to be able to target cells. of a given type and their ability to be endocytosed. They also allow the coupling of numerous ligands.

The reagents of the invention are therefore particularly useful, in particular, in gene cloning strategies.

They make it possible, in fact, to select the cells which have received a cloned or non-cloned gene or a nucleic acid sequence since the ligand attached to the liposome is capable of specifically recognizing a cell surface protein encoded by the gene or the nucleic acid sequence in question.

The invention thus provides the means of being able to isolate eukaryotic cells having received a given DNA sequence.

These reagents are also advantageously usable for the selection from a cell line of variant cells.

For this purpose, targeted liposomes are used using monoclonal antibodies which specifically recognize variant cells thanks to the high degree of selectivity of these liposomes.

Other characteristics and advantages of the invention are reported in the examples which follow and with reference to the boards on which:

FIGS. 1a to 1d represent the number of cells as a function of the days, of culture, under different conditions,

FIGS. 2a and 2b respectively illustrate cell growth and the percentage of fluorescent cells in the case of specific protection of a cell subpopulation and

- Figure 3 shows the curves obtained by measuring ¹²⁵ I of monoclonal antibodies linked to variant cells.

EXAMPLE I Preparation of Liposomes Containing THF Small unilamellar liposomes are prepared by sonication under nitrogen for 30 min at 50 ° C. as described by BARBET J. et al in Supramol. Struct. Cell Biochem. 16,243-258.

The liposomes are formed from 64 mol% of dipalmitoyl-phosphatidylcholine (product marketed by SIGMA) 35 mol% of cholesterol (marketed by FLUKA) and

and 1 mol% of dipalmitoyl phosphatidylethanolamine (marketed by CALBIOCHEM) modified with N-hydroxy-succinimidyl-3- (2-pyridyldithio) -propionate or SPDP (marketed by PHARMACIA). The amount of lipids is 40μmole.

The liposomes contain 100 mM of formyltetrahydrofolate (THF) and 20 mM of carboxyfluorescein (marketed by EASTMAN), or CF, in 100 mM of NAHCO ₃ . The THF obtained as a calcium salt is first passed through a column of Chelex

100 (Biorad) treated with HCl / 1N and 0.5N NAOH to obtain

sodium salt. Conventionally, THF and CF are passed over LH-20 columns (PHARMACIA) to remove hydrophobic contaminants.

- Iodization of S aureus protein A - Iodinate 100 μg of protein A (PHARMACIA) according to the method described by HUNTER and GREENWOOD in Nature 1962, 194, pages 495 to 497 with sodium iodide ¹²⁵ I (New England Nuclear) 0.5mCi and 7 μg of chloramine T then diluted with unlabeled protein A until a final specific activity of 5mCi / umoles.

- Coupling reaction between liposomes and protein A -

The liposomes are incubated with protein A modified by the SPDP (10 moles per mole of protein A) at a final protein A concentration of 180 to 200 μg / ml. The total lipid concentration is 7.5 mM. Under these conditions, 40 to 50% of protein A becomes linked to the liposome, which corresponds to averages of 15 to 20 molecules of protein A linked by liposomes of 500-600 Å in diameter.

After the coupling reaction, heat-inactivated fetal calf serum is added to a final concentration of 5% compared to the liposome preparation which is dialyzed for approximately 14 hours at 4 ° C. against a Hepes buffer (145 mM NaCl ; 10mM Hepes; pH 7.45).

Under these conditions, the losses in liposome content are 0.5 to 1% measured by fluorescence. After dialysis, it is sterilized by filtration on 0.45 μm millipore filters. The content of the liposomes remains encapsulated stably for several months at 4ºC.

These small liposomes can be stored indefinitely by freezing at -180 ° C. EXAMPLE 2 Use of liposomes according to Example 1 on which monoclonal antibodies are fixed for the positive selection of cells.

The results reported below relate to cultures of Ltk- cells (murine cells LH-2K ^k positive, HLA negative, thymidine kinase negative ⁾ , TRH 42 cells (L cells transfected with HLA and Herpes simplex thymidine kinase), of cells murine RDM4 tumors (H-2K ^k positive, HLA negative) and human fibroblasts A431 (H-2 negative, HLA-B, C positive). These cultures are carried out at 37 ° C. in 7% of C0 ₂ , 93% of air in tissue culture flasks with RPMI 1640 medium (Gibco) supplemented with 5% of heat-inactivated fetal calf serum (Eurobio). The antibodies used to target the cells are monoclonal antibody to mouse IgG2a, k, namely antibodies H100-5 / 28 15.5.5 and B1 23.2 .H100-5 / 28 has an affinity for the molecule H-2K ^k coded by the major murine incompatibility complex (MHC); 15.5.5 presents an affinity for the H-2D ^k molecules coded by murine MHC.

B1.23.2 is directed against the non polymorphic determinants expressed on the HLA-B and-C molecules coded by human MHC.

These antibodies are purified from culture supernatants of hybridoma cells on columns of Sepharose protein A (PHARMACIA).

. study of the specific action of liposemes containing THF vis-à-vis the proliferation of a given cell population. Murine cells Ltk-, TRH 42, murine tumor cells RDM4 and human fibroblast cells A431 are cultivated at 37 ° C. in 7% CO ₂ and

93% air on cell culture plates with RPMI 1640 medium (marketed by GIBCO) added with

5% inactivated fetal calf serum (marketed by EUROBIO). The cells are used at a rate of 5 × 10. ⁴ cells in 0.5 ml of medium.

Different test conditions were used to study the action of the liposomes of the invention. In FIGS. 1a to 1d, the results obtained are reported respectively with the cultures of TRH 42 (1a), Ltk- (1b), A431 (1c) and RDM4 (ld) cells.

On the curves given in these figures, the number of cells (x10 ^-5 ) is indicated on the ordinate and the number of culture days on the axis, each curve corresponding to the following culture conditions:

- with only the middle (curve 0),

- with only free Mtx (curve

),

- with free Mtx and liposomes containing THF, without Z antibody,

- with free Mtx and free THF

,or

- with free Mtx and liposomes containing THF, to which antibodies are fixed. anti-H-2K ^k ☐ or anti-HLA ☐,

The concentrations of Mtx in the culture medium, of free THF and of THF encapsulated in the liposomes are as follows according to the type of cells:

Concentration cells

Mtx THF THF in free encapsulated nM

TRH 42 500 4 μM 4 μM

Ltk- ""

A 431 250 4 μM 4 μM

RDM 62.5 2 μM 2 μM

The THF concentration used is determined for each type of cell by biological test using radio-labeled de oxyuridine. In these tests, we chose the minimum necessary concentration of THF, to eliminate the effect of Mtx on incor _¬ ration die oxyuridine.

Liposomes are used which contain 100 mM of THF. The THF concentration in the culture is obtained by diluting the liposomes by measuring the encapsulated CF with reference to standard CF of known concentration.

Final antibody concentrations are 4 to 8 µg / ml. The culture medium is changed

(daily) and the above culture conditions for each type of cell are again implemented two days later.

The cells are counted every day. The results obtained show that when the cells are exposed to quantities of free Mtx sufficient to kill them in the absence of THF, only cells expressing target determinants for THF-containing liposomes proliferate.

This proliferation is as good as with free THF. Thus, murine cells (Ltk-, TRH 42 and

RDM4) are not killed by the action of free Mtx when liposomes containing THF and carrying the anti-H-2K antibody are used.

On the contrary, human A431 cells are not protected and die.

When cells are incubated with anti-HLA antibody, only cells expressing HLA molecules, i.e., TRH42 and A431 cells, are not killed by free Mtx and proliferate as well as with free THF.

. Specific protection of a subpopulation of cells using liposomes containing THF and carrying an antibody.

The results obtained concerning the protection with respect to free Mtx in the culture medium of TRH 42 cells (HLA positive and H-2K ^k positive) are reported below in the presence of the anti-HLA monoclonal antibody. using liposomes carrying protein A containing THF. To this end, TRH42 and Ltk- cells are cultured, according to different reports, at a concentration of 10 cells in 0.5 ml of culture medium in the presence of 500 nM of Mtx and 4 μM of free or encapsulated THF.

Figures 2a and 2b respectively illustrate cell growth and the percentage of fluorescent cells.

The different curves correspond to the following respective conditions: curve

: use of free curved THF ☐: use of THF encapsulated in liposomes with curved anti-HLA antibody ▅: like the curve above but the antibody consists of anti-H-2K. Δ curve: without antibody

With the anti-HLA antibody, the cellular ratios of TRH42 / Ltk- are respectively 1/100 -curve—), 1/1000 (curve - - -); 1/10000

(curve -.-.-.) and 1 / 100,000 (......).

A TRH42 to Ltk- cellular ratio of 1/1000 with the anti-H-2K antibody is used as a control, as in the case of protection with free THF. During the protection with the antibodies, the cells are directly observed using a fluorescence microscope without other labeling, the liposomes containing CF co-encapsulated with THF.

As a control, aliquots of cells saved by free THF in the case of a cellular ratio of TRH42 to Ltk- of 1/100, are incubated with anti-HLA antibodies in the presence of fluorescent liposomes and observed under a fluorescence microscope .

After 3.7 and 11 days of culture, free Mtx (500nM) is added.

After 4.8 and 12 days, the medium is changed and the selection is repeated as for the first day.

At the end of the fifteenth day, when an active cell proliferation is observed, the medium is aspirated and Mtx is added to a final concentration of 1000nM.

No liposomes are added. After one more day, the medium is aspirated and an additional selection cycle is applied (liposomes with or without antibodies, plus 500nM of Mtx).

The cells are then grown in flasks, initially in the presence of 4 μM of free THF for 3 to 4 days and then in a normal medium.

During the positive selection, the cells targeted with the liposomes containing both THF and CF are observed directly under the fluorescence microscope and the fluorescent cells are counted.

The results obtained show that the cells carrying HLA are saved even in the case of cell ratios of 1 / 100,000 in TRH42 / Ltk- cells.

The targeted cells remain viable while the non-targeted cells are killed by free Mtx in the medium.

It is observed that when the cell population is targeted using the anti-H-2k antibody (which binds to all cells), all of the cells are protected from the action of Mtx by liposomes containing THF .

In contrast, when cell populations are incubated with liposomes without antibodies, all cells are killed. After the cell selection and growth operation, all specifically saved cells are examined under a fluorescence microscope after labeling with anti-HLA or anti-H2k antibodies or no antibody and liposomes containing CF. All cells appear with the HLA and H-2K molecules, but in the case of incubation with liposomes without antibodies, no cell is fluorescent. These results are confirmed by using another fluorescent anti-HLA antibody directed against an epitope of the HLA molecule. different from that retained by the antibody of the transfected HLA molecule used to select the cells. A sensitive radioimmunoassay using anti-HLA monoclonal antibodies labeled with 125I sodium iodide confirms these results.

EXAMPLE 3 Application of the Liposomes of the Invention in Gene Cloning Strategies -

DNA from a particular human species is inserted into host cells, in a manner known per se, into cells of a different species, for example from mice. The transfected cells which contain the desired gene or sequence are selected by the technique of the invention and their DNA is inserted, during a second transfection step, into recipient cells. The positive cells are then used to build a ban. in vectors (phage, plasmid or others).

This library can be reviewed with whole DNA from the donor species labeled on the basis of expression by those of the few vectors which have received the donor DNA which can hybridize. Positive vectors in hybridization are used to transfect recipient cells.

Restriction maps of positive transfection vectors can be used to determine the ends of the gene sought.

In an alternative cloning strategy, DNA is used to transfect cells and cells which express the desired gene are isolated by the technique of the invention. A second transfection step can be provided to reduce the amount of non-relevant DNA. The DNA of these cells is hybridized to the mRNA of cells not expressing the transfected gene.

Non-hybrid DNA is selected on hydroxyapatite columns. This DNA is radiolabelled and used to screen a genomic bank.

Colonies of positive vectors can serve as DNA donors to transfect other mammalian cells and confirm the presence of the gene and map its ends with restriction enzymes. . identification of vector having received a given DNA sequence using the liposomes of the invention. Whole cell DNA is used to build a library of approximately 200,000 vectors.

This library is grown in E. Coli or other suitable bacteria and then divided into 10 groups of 20,000 vectors each. The extracted DNA is used to transfect 10 wells containing recipient cells. In general, only one of these ten groups grows using the technique of the invention because only one (or two for a diploid genome) of the vector groups has received the DNA corresponding to the selected gene.

The wells in which positive growth is observed define the group of vectors which have received the gene.

This group of vectors is divided into 10 groups of 2000 vectors and repeated selection.

The positive well is then divided into 10 groups of 200 and after identification of the positive well into 10 groups of 20 for a final transfection.

The 20 vector clones in the positive well constitute a mapping for the restriction enzymes. If there is any ambiguity for the positive clone, its identity can be verified by transfection. This strategy is independent of the DNA donor species and can be used with mouse cells as a host for mouse DNA or human DNA. EXAMPLE 4 Application of the Liposomes of the Invention for the Selection of "Variant" Cells

The procedure is followed to isolate cells expressing H-2D ^k molecules at high rates. RDM4 cells are cultured, at a rate of 5 × 10 ⁴ cells in 0.5 ml of culture medium, in plates of groups of 24 wells, either with monoclonal antibodies capable of being recognized (anti-H-2D ^k ; 15.5.5), either with control antibodies (anti-HLA; B 1.23.2) and liposomes carrying protein A and containing THF.

Initially, we operate in the presence of 125nM. of free Mtx.

The concentration of THF encapsulated in the liposome is 100 mM.

When cell proliferation is observed, the medium is changed and the concentration of Mtx is doubled (at 250 nM) in the absence of liposomes, until observation of the arrest of cell growth.

At this time, the medium is aspirated and fresh medium is added with 2 μM da THF provided by the liposomes and antibodies as well as 125 nM of Mtx.

After 3 to 5 cycles of this selection, the cells are cultured with 125 nM of Mtx for 2 days and then incubated in a fresh medium for expansion.

The selected cells are then analyzed in a radioimmunoassay to check their capacity to bind to the monoclonal antibody which allows the selection to be made (15.5.5).

To carry out the radioimmunoassay, proceed as follows. Antibodies 15.5.5 and B.1.23.2 are labeled with 125I sodium iodide up to a specific activity of 100mCi per μmol as described above for protein A.

The unlabeled antibodies are added according to serial dilutions starting at 5 μg per well of microtiter plates comprising 96 conical wells with 3 × 10 ⁵ cpm of the same labeled antibody. 5 x 10 wild type RDM4 and RDM4 cells selected according to the high H-2D ^k expression are added to the wells in the culture medium.

After 2 hours of incubation at 4 ° C, the cells are washed 4 times by centrifugation and counted for ¹²⁵ I in a gamma counter. The results obtained are reported in FIG. 3 in which the dotted curve relates to the results obtained by measuring ¹²⁵ I with a RDM4 strain of wild type and the curve in solid lines to those obtained with RDM4 cells with high expression of H- 2D ^k .

The symbols ▅ correspond to the measurements carried out at different concentrations of antibodies 15.5.5 and anti-H-2D ^k . The curves with the symbols ⃞ correspond to the measurements carried out with the antibody B1.23.2 or anti-HLA.

Claims

1. Liposome compositions characterized in that they comprise at least one compound active with respect to cell proliferation, encapsulated in a liposome to which is linked a ligand capable of specifically targeting the cells whose survival is desired.

2. Compositions according to claim 1, characterized in that the active compound comprises or is formed of tetrahydrofolate or of a derivative, in particular N ⁵ formyl-tetrahydrofolate or also of a salt of tetrahydro-folate or of its derivatives.

3. Compositions according to claim 2, characterized in that the tetrahydrofolate or its derivatives are in the form of a salt, in particular a sodium or potassium salt.

4. Compositions according to any one of the preceding claims, characterized in that the ligand is chosen from monoclonal antibodies, lectins, hormones, protein-transports and in general any compound having an affinity for an expressed phenotype by the cell to be saved.

5. Compositions according to any one of claims 1 to 4, characterized in that they comprise liposomes preferably of less than 4000 Å, more especially less than 1000 Å, or even of dimensions less than 600 Å, approximately based phospholipids, advantageously conjugated or coupled by covalent bond to a compound making it possible to ensure the binding with the chosen ligand, the liposomes also contain, where appropriate, a marker allowing the identification of the targeted cells to which the liposomes have attached, such as carboxyfluorescein.

6. Compositions according to claim 5, characterized in that they contain an antibody mσnoclonal linked to protein A.

7.- Compositions according to any one of the preceding claims, characterized in that the liposomes are purified, for example on a chromatography column.

8.- Application of the compositions according to any one of claims 1 to 7, as biological reagents.

9. Biological reagents comprising at least one composition according to any one of claims 1 to 7 uriles in particular for targeting a given cell population and ensuring its survival.

10.- Use of a biological reagent according to claim 9 in clonago gene strategies or for the selection of variant cells.