WO1996002278A1

WO1996002278A1 - Immunonanoparticles coated with anti-beta-2 microglobulin monoclonal antibodies

Info

Publication number: WO1996002278A1
Application number: PCT/FR1995/000960
Authority: WO
Inventors: Nicole Bru-Magniez; Jean-Claude Chermann; François LESCURE; Jean-Marie Teulon; Pascal Breton; Xavier Guillon
Original assignee: Laboratoires Upsa; Institut National De La Sante Et De La Recherche Medicale
Priority date: 1994-07-18
Filing date: 1995-07-18
Publication date: 1996-02-01
Also published as: AU3079895A; FR2722411B1; FR2722411A1

Abstract

Immunonanoparticles consisting of nanoparticles of a polymeric methylidenemalonate compound coated with anti-β2 microglobulin antibodies, drugs containing same, and the use thereof for preventing and/or treating diseases caused by HIV infection, for making drugs for preventing and/or treating such diseases, as biological reactants, or in a method for screening for the presence of β2 microglobulin in free form or combined with viral particles in a biological fluid sample.

Description

Immunonanoparticles coated with anti-beta-2 microglobulin ine monoclonal antibodies

Immunonanoparticles coated with anti-β2 microglobulin monoclonal antibodies, medicaments containing them and their use for the prophylaxis and / or treatment of pathologies due to infection by the HIV virus.

The invention relates to immunoparticles consisting of polymerized methylidene malonate nanoparticles coated with anti-β2 microglobulin antibodies, the drugs containing them, their use for prophylaxis and / or the treatment of pathologies due to infection by the HIV virus, for the preparation of drugs containing them or as biological reagents. The> 2 microglobulin (hereinafter also called β2m) is a polypeptide constituting the light chain of HLA (human leukocyte antigen) class I antigens. Β2m is associated with the heavy chain of HLA class I antigens.

The association of cellular proteins such as β2 with the viral envelope has been reported by L. Arthur et al, Science, 1992, 2 J 1935-1938.

The ability of anti-β2 microglobulin (anti-β2) monoclonal antibodies to delay the production of HIV 1 virus in blood, peripheral mononuclear cells (PBMC) was reported in the publication P.Corbeau et al, J. Viral. , 1990,, 1459-1464. The delay in the appearance of the cytopathogenic effect of the HIV 1 virus vis-à-vis MT4 cells by anti-β2m monoclonal antibodies is also described in C. Devaux et al, Res. Immunol., 1990, 141.357-372.

We have now found that it is possible to use immunonanoparticles carrying anti-β2 microglobulin monoclonal antibodies capable of specifically recognizing β2 microglobulin, in particular when it is combined with viral particles for the prophylaxis and / or treatment of pathologies. due to infection with the HIV virus, especially AIDS.

By "β2 microglobulin" is meant in the following description the β2 microglobulin alone or associated with the heavy chain of HLA class I antigens.

The immunonanoparticles according to the invention have the advantages of conferring on said antibodies better stability, greater activity and better immunoreactivity. They are also completely biodegradable and have very low toxicity, their degradation products being themselves non-toxic. In addition, they can advantageously be sterilized and lyophilized.

The invention therefore relates to immunonanoparticles consisting of nanoparticles of polymer of a methylidene malonate compound of formula (I):

COORi H ₂ C = C ^ (I)

COO- (CH ₂ ) n-COOR ₂

in which R ^ and R2, identical or different, represent a linear or branched C1-C5 alkyl group and n is an integer from 1 to 5, said nanoparticles being coated with anti-β2 microglobulin antibodies capable of specifically recognizing β2 microglobulin .

According to one aspect of the invention, said immunonanoparticles consist of nanoparticles as defined above coated with anti-β2 microglobulin antibodies capable of specifically recognizing free β2 microglobulin, or associated with viral particles or cells.

In a preferred aspect, said immunonanoparticles consist of nanoparticles as defined above coated with anti-β2 microglobulin antibodies capable of specifically recognizing β2 microglobulin when it is associated with viral particles.

The preparation of the compounds of formula (I) is described in patent EP 0283364.

The compounds of formula (I) polymerize in the form of a polymer network to form the nanoparticles having an average diameter preferably between 100 and 500 nm, more particularly between 100 and 400 nm, in particular of the order of 300 nm or inferior. The molecular weight by weight (Mw) of the methylidene malonate polymers of formula (I) can be of the order of at least 1500 to 2000, in particular between 1500 and 50000, expressed in polystyrene equivalents.

Preferably, the methylidene malonate compound of formula (I) is l-ethoxycarbonyl-l-ethoxycarbonylmethyleneeneoxycarbonylethene (compound of formula (I) in which R \ = R2 = ethyl and n = 1). The immunonanoparticles according to the invention can advantageously contain within their polymer network a biologically active substance, preferably an anti-viral agent. As examples of anti-viral agents, non-limiting mention may be made of didanosine, ribavirin, zidovudine, aciclovir, vidarabine and zalcitabine.

In an advantageous aspect, the monoclonal anti-β2 microglobulin antibody with which the nanoparticle is coated to constitute the immunonanoparticle is chosen from murine antibodies B1.1G6, B2.62.2 (IMMUNOTECH., France) and BBM 1 (Institut Pasteur, France ), the antibody B1.1G6 being preferred. Monoclonal antibodies will in particular be used in the form of a solution containing bovine serum albumin as supplied commercially.

The anti-β2 ^m monoclonal antibodies are fixed on the nanoparticles to constitute the immunonanoparticles by techniques known per se, preferably by passive adsorption, in particular in a phosphate buffer.

Said antibodies can also be attached to the nanoparticles by specific adsorption in particular via protein A of Staphylococcus aureus or a biotin-avidin bond.

Different methods of fixing antibodies are described in particular in the publication L. Illum et al, Meth. Enzymol., 1985, H2, 67-84.

In another aspect, the invention relates to a medicament containing the immunonanoparticles described above as active principle, in association with a pharmaceutically acceptable vehicle.

In the medicaments according to the present invention for oral, sublingual, subcutaneous, intramuscular, intravenous, transdermal, local or rectal administration, the active ingredients can be administered in unit administration forms, in admixture with conventional pharmaceutical carriers, animals and humans. Suitable unit administration forms include oral forms such as tablets, capsules, powders, granules and oral solutions or suspensions, sublingual and oral administration forms, subcutaneous administration forms , intramuscular, intravenous, intranasal or intraocular and forms of rectal administration. In a further aspect, the invention relates to the use of the immunonanoparticles described above for the prophylaxis and / or the treatment of pathologies due to infection with the HIV virus, and in particular for the manufacture of a medicament for prophylaxis and / or the treatment of pathologies due to infection with the HIV virus.

Indeed, the immunonanoparticles described above have properties of inhibiting the formation of syncytia in the experimental model of F. Rey et al, Virology, 1991, Ifi 165-171.

According to another aspect, the invention also relates to a method for detecting the presence of free β2m or associated with viral particles in a sample of biological fluid originating from a mammal, human or animal, consisting in bringing a sample of this biological liquid with immunonanoparticles described above, consisting of nanoparticles of polymer of a methylidene malonate compound of formula (I): COORi

H ₂ C = C ^ (I)

^X COO- (CH ₂ ) _n -COOR ₂ in which Rj and R2, identical or different, represent a linear or branched C1-C5 alkyl group and n is an integer from 1 to 5, said nanoparticles being coated with anti -β2m capable of specifically recognizing the free 2m or associated with viral particles, in sufficient quantity to form an antigen-antibody complex capable of being detected.

In a preferred aspect, said method will be used for the detection of the presence of β2m when it is associated with viral particles, the immunonanoparticles according to the invention being made up of nanoparticles as defined above coated with anti-β2m antibodies likely to specifically recognize β2m when associated with viral particles.

The detection of free β2m is also of interest since it has been described that the level of free β2m increases with the course of the disease in seropositive patients (JL Fahey et al., New Engl. J. of Medicine , 1990, 222, 166-172).

In another aspect, the invention also relates to the use of the immunanoparticles coated with anti-β2m antibodies described above as biological reagents, in particular as controls for the evaluation of the activity of compounds in a model of inhibition of the formation of syncytia, or also in techniques of immunoseparation, immunoprecipitation, immunostaining or immunopurification . The invention is illustrated by the examples below which are in no way limiting.

EXAMPLE 1 Preparation of immunonanoparticles of ethoxycarbonyl-1-methyleneoxycarbonyl-1-ethene coated with anti-β2 microglobulin B1.1G6 antibodies. n Preparation of nanoparticles:

The nanoparticles are prepared under sterile conditions at 25 ° C. by emulsion-polymerization of the monomer as described in F. Lescure et al, Proceed. Intem. Nice. Control. Rel. Bioact. Mater, 1991, 18, 325-326, or F. Lescure et al. Pharm. Res., 1994 J 1270-1277.

Briefly, 100 l of ethoxycarbonyl-1-methyleneoxycarbonyl-1-ethene monomer (UPSA Laboratoires / CARPIBEM, France) are depleted in SO2 under vacuum for 3 h at 10 ^~ 2 torr, then added dropwise with stirring to a medium. polymerization (10 ml) thermostatically controlled at 25 ° C consisting of a KH ₂ Pθ4 / NaH2PO4 buffer, 0.066 M, pH 5.5 containing 1% dextran (M _r = 70,000,

FLUKA CHEMIE, Switzerland).

The whole is left under stirring for 18 h then the nanoparticles are recovered after filtration on 8 m filter paper (Whatman, Great Britain), then divided into aliquots, lyophilized and stored at room temperature protected from light.

At the time of use, the nanoparticles are resuspended in sterile ultrafiltered water so as to obtain a polymer suspension substantially equal to 10 m g / ml. The average size of the nanoparticles is measured before and after lyophilization using a device for analyzing submicrometric particles (Coulter

Electronics, USA). The average particle diameter is around 320 nm. The molecular masses of the constituent polymers of the nanoparticles are determined by steric exclusion chromatography (SEC) (Polymer laboratories, Great Britain). For analysis, the nanoparticulate suspension is ultracentrifuged at 200,000 g for 5 minutes, then the pellet is taken up in THF to which 0.5% toluene (internal standard) is added. The solution thus obtained is filtered and 20 μl of the filtrate are injected for analysis. Detection is done by refractometry. 2 ^ Attachment of antibodies to nanoparticles

The antibody B1.1G6 (Immunotech, France) in lyophilized form is taken up in ultrafiltered water so as to obtain a solution containing 250 μg / ml of antibody and 0.125% bovine serum albumin (BSA). 200 μl of the antibody solution are taken, to which 30 μl of the suspension of nanoparticles are added: the mixture is adjusted to 250 μl with phosphate buffer pH 7.4. A suspension is thus obtained containing 1.2 mg / ml of nanoparticles, 200 μg / ml of antibody B1.1G6 and 0.1% of BSA. The mixture is incubated at 20 ° C for 2 hours with shaking. One can also make a suspension containing 100 μg / ml of B1.1G6 antibody and 0.05% of BSA for 1.2 mg / ml of nanoparticles.

EXAMPLE 2 Determination of the rate of binding of the antibody B1.1G6 and study of the stability of the immunonanoparticles of example 1. Fixation and assay of the antibodies B1.1G6 The nanoparticles as obtained in example 1, step 1 ) are diluted in a pH 7.4 buffer solution containing anti-β2 microglobulin B1.1G6 antibody (stock solution at 100 μg / ml, 0.2% bovine serum albumin, IMMUNOTECH, France). 3 suspensions containing 5 are produced; 2.5 or 1.25 μg / ml of antibody for 2.5 mg / ml of nanoparticles, which are incubated at room temperature for 2 h with shaking.

After the incubation (T = 0), the free antibody is separated from the immunonanoparticles by ultracentrifugation at 40,000 g for 5 minutes. 1 \ Determination of the rate of binding of the Bl .1G6 antibody by ELISA assay a) The antigen, β2 microglobulin (SIGMA, United States) is dissolved in a carbonate / bicarbonate buffer pH 9.6 at the concentration 2.5 μg / ml. This solution is distributed on a 96 well IMMULON III plate (DYNATECH, France), at a rate of 100 μl per well. The plate containing the antigen is incubated overnight at + 4 ° C. The wells are then emptied, and 125 μl of blocking solution (PBS buffer containing 0.5% BSA and 0.001% sodium azide) are added per well. The system is incubated for 1 hour at 37 ° C.

The wells are then emptied, then rinsed 4 times with the washing solution (PBS buffer 0.5% Tween 20). b) The solutions containing the anticoφs B1.1G6 (fixed anticoφs to be assayed as well as the antibody solutions constituting the calibration range) are distributed in the wells at the rate of 50 μl per well.

- Preparation of the fixed anticoφs solution to be assayed: At the end of the nanoparticle-anticoφs incubation step, the suspension is subjected to ultracentrifugation; the anti∞φs not fixed and separated from the immunoparticles is collected with the supernatant in which it will be assayed.

- Constitution of the calibration range:

The calibration range comes from a stock solution produced in an identical fashion to the suspension of immunonanoparticles, but where the nanoparticles are replaced by an ultracentrifugation supernatant containing polymerization medium and soluble oligomers. This mixture is treated under the same incubation conditions as the nanoparticle-antibody suspension.

The calibration range is carried out by serial dilution to half the stock solution.

The system is incubated for 1 hour at 37 ° C.

The wells are then emptied, then rinsed 4 times with the washing solution. c) 50 μl of the working solution are then added (2 μl of polyclonal anticoφs of sheep anti-mouse Fc fragment (SIGMA, France) labeled with alkaline phosphatase, in 16 ml of washing solution).

The system is incubated for 1 hour at 37 ° C.

The wells are then emptied, then rinsed 4 times with the washing solution. The substrate solution (0.1 M glycine buffer solution at pH 10.4 containing 1 mg / ml of -nitrophenylphosphate) is then introduced at the rate of 100 μl per well.

The plate is then incubated at 37 ° C. under 5% of b. After approximately 2 hours of incubation, the assay is carried out by colorimetric reading at 405 nm using a MICROPLATE EL-310 plate reader (BIOTECH INSTRUMENT, United States ). The results are reported in Table 1 below:

Table 1

* the concentrations of anticoφs are expressed in μg / ml and for 2.5 mg / ml of nanoparticles. 3) Determination of the stability of immunonanoparticles

The suspension of immunonanoparticles at 5 μg / ml of anticoφs B1.1G6 for 2.5 mg / ml of nanoparticles is stored at 37 ° C. for 48 or 72 hours. At the end of each period, the suspension is ultracentrifuged and the free anticoφs collected in the supernatant is assayed by ELISA assay as above (T = 48 h and 72 h). The anticoφs solution used in the calibration of the ELISA assay undergoes the same treatment as the suspension studied. The stability of the system is evaluated according to the variation in the fixation values which is observed during this period.

The results are reported in Table 2 below:

Table 2

Percentage of fixation

Initial After 48 h After 72 h of incubation incubation

Percentage of anticoφs fixed 50 ± 5 42 ± 5 41 ± 5 on nanoparticles

Quantity of anticoφs fixed, in l ± Ol 0.84 ± 0.1 0.82 ± 0.1 μg / mg of polymer The results show that the percentage of fixed anticoφs decreases by 20% during the 48 hours which follow the formation of the immunonanoparticles but that a state of stability is then reached.

EXAMPLE 3 Study of the inhibitory activity of the immunonanoparticles of Example 1 with respect to the formation of syncytia in an in vitro model. a) The freshly prepared immunonanoparticles are diluted 2 / 15th in RPMI 1640 (GIBCO, France) supplemented with 10% fetal calf serum (SVF), 1% L-glutamine (L-Gln) and 1% NHP antibiotics (Penicillin, Streptomycin, Neomycin). b) The anticoφs B1.1G6 in lyophilized form is taken up in ultrafiltered water so as to obtain a solution containing 250 μg of anticoφs and 0.125% of BSA in PBS.

A standard range of anticoφs B1.1G6 is prepared by dilution using RPMI 1640 medium supplemented with 10% of SVF fetal calf, 1% of L-Gln and 1% of NHP antibiotics. c) Pre-incubation for 2 h at 37 ^# C 100 μl of a 2.10 ^-3 dilution of the HIV-1 virus from supernatants of infected cells (CD4 ⁺ cells, for example MT4, cultured with HIV-1 virus strain BRU ), this dilution having been determined to induce the formation of syncytia in 4 to 6 days, with:

- either 100 μl of suspension of immunonanoparticles or nanoparticles,

- either 100 μl of dilution of anticoφs (standard range prepared from 80 μl of the stock solution of anticoφs, supplemented with phosphate buffer pH 7.4),

- or 100 μl of culture medium for the cell and virus controls. At the end of this time, the infection is carried out in a 96-well microplate by adding the 200 μl previously obtained, to a pellet of 3.10 ^ MT4 cells. After an incubation of 1 hour at 37 ° C., the MT4 cells are washed 3 times with unsupplemented RPMI 1640, then cultured at a concentration of 3.10 4 cells per ml in 24-well microplates in RPMI 1640 at 10% SVF , 1% L-Gln, 1% PSN.

Every 3 or 4 days, the cells are passed and adjusted to the concentration of 3.10 ⁵ cells per ml with RPMI 1640 medium at 10% FCS, 1% L-Gln, 1% PSN. After 4 days of culture, the appearance of syncytia is observed under the microscope every 24 hours (2 measurements) in parallel with the following controls:

- nanoparticle control (nanoparticles alone + uninfected MT4 cells),

- virus control (infected MT4 cells, without immunonanoparticles), - cell control (uninfected MT4 cells, without immunonanoparticles).

The results are reported in Tables 3 and 4 below, in which the formation of syncytia is noted in the following manner: (+) onset of appearance of syncytia (we observe only a few) + presence of syncytia in more appreciable quantity

++ presence of many syncytia

T toxic (dead cells).

Table 3

Total concentration in Concentration in day 4 day 5 day 6 day 7 antibody (μg / ml) polymer (μg / ml) immunonano¬ 3.3 20 - - (+) (+) ++ ++ ++ T ++ T particles coated 1.6 10 - (+) (+) (+) ++ ++ ++ T ++ T of antibody B1.1G6 0.8 5 - (+) + + ++ ++ ++ T ++ T

0.4 2.5 (+) (+) + + ++ ++ ++ T ++ T

antibody B1.1G6 100 - - - - - - - - free 50 - - - - (+) (+) ++ ++

25 - - - (+) (+) + ++ T ++ T

12.5 (+) (+) + + ++ ++ ++ T ++ T

6.25 (+) (+) + + ++ ++ ++ T ++ T

3.13 iϋ. (+) + + ++ ++ ++ T ++ T control 80 - - - - - - nanoparticles cell control - - - - - - - - virus control Si (+) + + ++ ++ ++ TT

The results show that by using the immunonanoparticles according to the invention, an inhibition of the formation of syncytia is obtained from a concentration of anticoφs much lower than that of the free anticoφs. In particular, there is an inhibition on the 4th day from an antico ants concentration of 3.3 μg / ml for the immunonanoparticles according to the invention instead of 25 μg / ml of free anticoφs (Table 3).

On day 7, the same inhibition is observed, with an anticoφs concentration of 13.3 μg / ml for the immunonanoparticles according to the invention, as with 100 μg / ml of free anticoφs (Table 4).

The immunonanoparticles described therefore make it possible to lower the minimum effective concentration of anticoφs by approximately a factor 8 both on day 4 and on day 7 after infection, as can be estimated by the ratio of minimum effective concentration in free anticoφs / anticoφs linked to nanoparticles.

EXAMPLE 4

The inhibitory activity of the immunanoparticles of Example 1 as well as of the immunanoparticles coated with another antico ants anti-β2m, the anticoφs B2.62.2 (I munotech, France), obtained under the same experimental conditions as those of Example 1, with regard to the formation of syncytia using MT4 cells infected with a more cytopathogenic layer of the HTV-1 virus (HIV-1 NDK, Zairian strain), the other operating conditions being the same as those from example 3.

The results are reported in Table 5 below:

The results show that the inhibitory activity with respect to the formation of the syncytia of the immunonanoparticles according to the invention is comparable to that of Example 3 although the strain of virus HIV-1 used is more cytopathogenic.

Indeed, the ratio of effective minimum concentration in free anticoφs / effective minimum concentration in anticoφs linked to nanoparticles is greater than 8 for the 2 anticoφs tested.

EXAMPLE S;

We carried out a comparative study of the inhibitory activity of the formation of syncytia of immunonanoparticles coated respectively with anti-βom anticoφs B1.1G6, B2.62.2 (Immunotech, France) and BBMl (Institute

Pasteur, France) under the experimental conditions of Example 3 (MT4 cells infected with an HIV-1 virus strain BRU).

The results are reported in Table 6 below:

Table 6

Concentration in Concentration in day 4 day 5 day 6 day 7 antibody (μg / ml) antibody (μg / ml) immunonano¬ 13.3 80 (+) + (+) ++ particles coated with anticoφs B1.1G6

free antibody 100 B1.1G6 (+) + ++ immunonano¬ 13.3 80 (+) + particles coated with anticoφs B2.62.2 free antibody 100 (+) + (+) B2.62.2 immunonano¬ 13.3 80 ( +) + (+) ++ particles coated with antibody BBMl free antibody 100 + (+) ++ BBMl control 80 nanoparticles control cells control virus <+> + + ++ ++ ++ T ++ T

The results show that with the 3 anticoφs tested, a minimum effective concentration in free anticoφs / minimum effective concentration in anticoφs linked to the nanoparticle is obtained of the order of 8.

EXAMPLE 6

The comparative study of the inhibitory activity on the formation of syncytia of the immunonanoparticles of Example 1 coated with anticoφs was carried out.

B1.1G6 with isohexylcyanoacrylate nanoparticles (PIHCA) as described in patent EP 0 007 895, coated with the same anticoφs, under the experimental conditions of Example 3.

The results are reported in Table 7 below:

The results show that by using nanoparticles made up of a polymer different from that of the invention, one obtains from the 5th day a cellular toxicity not allowing by obtaining the advantageous inhibiting effect of the immunonanoparticles according to the invention.

Claims

1. Immunonanoparticles consisting of nanoparticles of polymer of a methylidene malonate compound of formula (I):

COORχ H ₂ C = C ^ (I)

^X COO- (CH ₂ ) n-COOR ₂

in which R \ and R2, identical or different, represent a linear or branched C1-C5 alkyl group and n is an integer from 1 to 5, said nanoparticles being coated with anti-β2 microglobulin anticoφs capable of specifically recognizing β2 microglobulin .

2. Immunonanoparticles according to claim 1, characterized in that said nanoparticles are coated with anti-β2 microglobulin anticoφs capable of specifically recognizing free β2 microglobulin, or associated with viral particles or cells.

3. Immunonanoparticles according to one of claims 1 or 2, characterized in that the methylidene malonate compound is a compound of formula (I) in which R = R2 = ethyl and n = 1.

4. Immunonanoparticles according to one of claims 1, 2 or 3, characterized in that the monoclonal anticoφs anti-β2 microglobulin are chosen from the anticoφs B1.1G6, B2.62.2 and BBMl.

5. Immunonanoparticles according to any one of claims 1 to 4, characterized in that the anti-monoclonal anti-β2 microglobulin is the anticoφs Bl.lGό.

6. Immunonanoparticles according to any one of claims 1 to 5, characterized in that the monoclonal anticoφs anti-β2 microglobulin are fixed on the nanoparticle by passive adsorption.

7. Immunonanoparticles according to any one of claims 1 to 6, characterized in that the nanoparticle has a mean diameter preferably between 100 and 500 nm, more particularly between 100 and 400 nm, in particular of the order of 300 nm or inferior.

8. Immunonanoparticles according to any one of claims 1 to 7, characterized in that the polymer of methylidene malonate compound of formula (I) has a molecular weight by weight (Mw) of the order of at least 1500 to 2000, in particular between 1500 and 50000, expressed in polystyrene equivalents.

9. Immunonanoparticles according to any one of claims 1 to 8, characterized in that the nanoparticles contain a biologically active substance, preferably an antiviral agent.

10. A drug containing immunonanoparticles according to any one of claims 1 to 9 as active ingredient, in combination with a pharmaceutically acceptable vehicle.

11. Use of immunonanoparticles according to any one of claims 1 to 9 for the prophylaxis and / or treatment of pathologies due to infection by the HIV virus.

12. Use of immunonanoparticles according to any one of claims 1 to 9 for the manufacture of a medicament for the prophylaxis and / or the treatment of pathologies due to infection by the HIV virus.

13. Use of immunonanoparticles according to any one of claims 1 to 9, as biological reagents.

14. A method of detecting the presence of β2 microglobulin free or associated with viral particles in a sample of biological fluid from a mammal, human or animal, consisting in bringing a sample of this biological fluid into contact with immunonanoparticles according to l 'any one of claims 1 to 9, in an amount sufficient to form an antigen-anticoφs complex capable of being detected.