CZ276295A3 - Immune nanoparticles carrying anti-cd-4 monoclonal antibodies and the use thereof for prophylaxis and/or treating pathologic states caused by infection with hiv viruses or as biological reaction substances - Google Patents

Abstract

The invention relates to immunonanoparticles comprised of nanoparticles of a polymer of a methyliden malonate compound coated with anti-CD4 antibodies, as well as their utilisation for prophylaxy and/or the treatment of pathologies due to an infection by the HIV virus or as biological reactants, or in the process for the detection of anti-CD4 antigenes in a biological mammalian liquid.

Description

Qblasttechrriky

The invention relates to immunoparticles consisting of nanoparticles (or microscopic particles) of polymerized methylidene malonate which are coated with anti-CD4 antibodies and their use for the prophylaxis and / or treatment of pathological conditions caused by infection with HIV viruses or as biological reactants.

BACKGROUND OF THE INVENTION

Numerous publications have been reported in the art to point out the importance of the role of CD-4 antigen in lymphocyte infection with human immunodeficiency virus (HIV) and its use as cellular HIV receptor receptors (see D. Klatzman et al. , Nature, 1984, 312, 767-786).

The ability of anti-CD4 monoclonal antibodies to inhibit the formation of syncytia (which are gigantic cells resulting from the pooling of healthy T lymphocytes and infected T lymphocytes) is described in F. Rey et al., Virology, 1991, 181, 165-171.

Conjugates of nanoparticles and monoclonal antibodies in which said nanoparticles consist of hexylcyanoacrylate polymer and monoclonal antibodies are described in prior art publications on the one hand as anti-alpha-fetoprotein antibodies (applied in alpha-fetoprotein assays) or on the other as antisarcoma antibodies (applied as antitumor agents); for example, C. Kubiak et al., Int. J. Pharmaceutics, 1988, 41, 181-187, respectively. L. Illum et al., J. Pharmacol. Exp. Therapeutics, 1984, 230, 733-736. However, these cyanoacrylic polymer-based conjugates exhibit toxicity to T4 lymphocytes.

Liposoray coated with anti-CD4 LEU3-A antibodies (or as carriers for such antibodies) are described in N. Phillips et al., Cancer Detect. Prev., 1990, 14, 383-390. However, these liposomes have the disadvantage of causing anti-liposome antibodies in mice (see N. Philipps, Second European Symposium on Controlled Drug Delivery, 1992, Northern Netherlands, The Netherlands, Abstract book, pp. 172-175) .

SUMMARY OF THE INVENTION

It has been found that immunoparticles carrying anti-CD4 monoclonal antibodies (or as carriers for such monoclonal antibodies) can be used for the prophylaxis and / or treatment of pathological conditions caused by human immunodeficiency viruses (or HIV viruses), particularly AIDS.

These immunoparticles have the advantage of providing improved stability, greater potency and improved immunoreactivity for said antibodies. These immunoparticles are also completely biodegradable and have very low toxicity, and their degradation products as such are also non-toxic. In addition, it can be advantageously sterilized and lyophilized.

The present invention relates to immuno-nanoparticles consisting of nanoparticles (or microscopic particles) of a polymer of a methylidene malonate compound of formula I

COOR-, /

h ₉ c = c \

C00- _{(CH2) 2} -COOR (I) wherein:

Rl and _R2, which may be identical or different, represent a linear or branched alkyl group having 1-5 carbon atoms, n is a number 1 to 5, coated with anti-CD4 antibodies.

The preparation of these compounds of formula I is described in detail in European Patent No. 2,283,364.

The compounds of formula (I) are also polymerized in the form of a polymer network to form nanoparticles having a diameter in the range of 100 nm to 400 nm, preferably 300 nm in diameter. The average molecular weight (Mw) of these methylidene malonate polymers of formula I is in the region of 600, expressed in polystyrene equivalents.

Preferably, the methylidene malonate compound is of formula (I)

1-ethoxycarbonyl-1-ethoxycarbonylmethyleneoxycarbonylethene, i.e. a compound of formula (I) above wherein R ₁ = R ₂ = ethyl and n is 1.

The immunoparticles of the invention may preferably contain biologically active agents, preferably antiviral agents, in their polymeric network structure. . reagents. Examples of such antiviral agents include, but are not limited to, didanosine, ribavirin, zidovudine, aciclovir, vidarabine, and zalcitabine.

In a preferred embodiment, the anti-CD4 monoclonal antibodies by which said nanoparticles are coated to form immunoparticles are selected from the group consisting of 0KT4 and 0KT4-A antibodies and 10T4a antibodies derived from a 13B8-2 hybridoma, preferably by these anti-CD4 monoclonal antibodies 10T4-α antibodies. These monoclonal antibodies are used in particular in the form of a solution containing bovine serum albumin, in the form in which it is commercially available.

Furthermore, anti-CD4 monoclonal antibodies labeled with a fluorescent substance such as fluorescein isothiocyanate may also be used, but unlabeled anti-CD4 antibodies are preferred.

These anti-CD4 monoclonal antibodies are bound to nanoparticles to form immuno-nanoparticles using conventional methods known in the art to bind said antibodies to the nanoparticles, preferably by passive adsorption, especially in phosphate buffer.

These antibodies can also be bound to nanoparticles by specific adsorption methods, in particular via protein A from Staphylococcus aureus or via biotin / avidin binding,

Various methods of binding antibodies have been described in the prior art, particularly L. Illum et al., Meth. Enzymol., 1985, 112, 67-84.

The invention also relates to the use of said immunoparticles according to the invention for the prophylaxis and / or treatment of pathological conditions caused by HIV virus infection, in particular their use for the preparation of medicaments for the prophylaxis and / or treatment of pathological conditions caused by HIV virus infection.

The immunoparticles of the invention described above exhibit the ability to inhibit syncytium formation according to the experimental model described by F. Rey et al., Virology, 1991, 181, 165-171.

The present invention also provides a method for detecting the presence of CD4 antigens in a biological fluid sample derived from a human or mammal animal by contacting the biological fluid sample with the aforementioned immunoparticles comprising the polymer nanoparticles of a methylidene malonate compound of the formula I:

COOR ₁ /

h ₉ c = c \

COO- (CH _{2) n} -COOR ₂ (I) wherein:

Rl and _R2, which may be identical or different, represent a linear or branched alkyl group containing 1-5 carbon atoms and n is a number from 1 to 5, wherein said nanoparticles are coated with anti-CD4 antibody in an amount sufficient to form antigen complex - an antibody capable of detection.

The invention also encompasses the use of the aforementioned immunoparticles coated with anti-CD4 antibodies as biological reagents, particularly as control agents for evaluating the efficacy of compounds in a model for inhibiting syncytium formation, or otherwise for the immunoseparation method, immunoprecipitation method, immunolabeling method and the like.

DETAILED DESCRIPTION OF THE INVENTION

Immunoparticles bearing anti-CD4 monoclonal antibodies and their use for the prophylaxis and / or treatment of pathological conditions, and the process for their preparation and properties will be described in the following by way of non-limiting examples.

Example 1

Preparation of 1-ethoxycarbonyl-1-ethoxycarbonylmethyleneoxycarbonylethene immuno-nanoparticles coated with anti-CD4 10T4a antibodies.

(1) Preparation of nanoparticles:

In this embodiment, nanoparticles were prepared under sterile conditions at 25 ° C by the monomer emulsion polymerization method described, for example, in F. Lescure et al., Proceed. Intern. Symp. Control. Rel. Bioact. Mater., 1991, 18, 325-326, Controlled Release Society Inc. Briefly, SO _{2 is} removed from 100 gL of 1-ethoxycarbonyl-1-methyleneoxycarbonylethene monomer (UPSA Laboratoires / Carbipem, France) under vacuum for 3 hours at 1 mbar. then added dropwise with stirring into the polymerization medium (10 ml) which is controlled at 25 C and which consists of 0.066 M KH ₂ P0 ₄ / NaH ₂ P04 buffer, pH 5.5 containing 1% dextran (M _r = 70,000, Fluka Chemie, Switzerland).

The mixture was stirred for 18 hours, then the nanoparticles were collected by filtration on 8 gm filter paper (Vhatman, UK) and aliquoted, which were lyophilized and stored at room temperature.

In use, these nanoparticles were resuspended in water for the preparation of injectables.

The mean size of these nanoparticles was measured before and after lyophilization using a submicron particle analyzer (Coulter Electronics, USA). This average particle diameter was found to be in the region of 320 nm.

The molecular weight of the component polymers of these nanoparticles was determined by steric exclusive chromatography (SEC) (Polymer Laboratories, UK). For analysis, the nanoparticle suspension was subjected to ultra centrifugation at 200,000 g for 5 minutes, and the residue was taken up in THF, to which 0.5% toluene was added, for injection. Detection was performed by refractometric method.

(2) Binding of antibodies to nanoparticles.

The nanoparticles were diluted to 2.7 mg / ml in phosphate buffer pH 7.4.

100 μ 450 of I0T4a monoclonal antibodies, which need not be labeled or labeled with fluorescein isothiocyanate (clone 13B8-2, 100 µg / ml solution containing 0.2% bovine serum albumin; Immunotech, France) were then added to the 450 µΐ suspension. The mixture was incubated at 20 ° C for 2 hours with stirring.

Example 2

Test for Stability of Immunoparticles according to Example 1.

In an xohoxo example, 50 gg / ml polymer conjugate immunoassays were added to RPMI 1640 phenol red-deficient media (GIBSCO, USA) and containing 1% gluxamine, 1% penicillin, 1% sxrepxomycin, 1% neomycin, and 10% fexal gel.

Proxilax coated nanoparticles were separated from the free proxilaxes by ulxraods by dilution at 40,000 g for 5 minux, both immediately and 2 hours after initiation of the 37 ° C xeplox incubation.

Isohexyl cyanoacrylax nanoparticles (PIHCA) were also prepared for comparison, with the preparation process being found in European Paxenx No. 0 007 895 and in United Paxenx No. 4 329 332. In the xomxo case, the nanoparticles were separated from the free proxilaxes. 80,000 g, performed for 5 minux.

MnožsXví bonds proxiláxek I0T4a was sxanoveno measuring the fluorescence of the supernatant (λ _exc = ^nm ix _ace 'λ emission ^{= NR} ^ ^for P and ^dip spekxrofluorimexru L530 (Perkin

Elmer Lxd, United Kingdom).

The concen- tration of free proxilaxes was calculated according to a calibration curve and the percentage of proxilax binding was estimated from the difference.

The results obtained with xomxo xesx are shown in the following xabules 1 and 2, where:

- in Table 1, the values obtained with the immuno-nanoparticles of Example 1 are shown, and

- Table 2 shows the results according to the comparative example, i.e. results with PIHCA nanoparticles coated with the same antibodies as the immuno-particles according to Example 1.

TABLE 1

Concentration antibodies Percentage share of bonds (gg / ml) In the begining After 2 hours incubation 0.1 24 ± 1.52 23 ± 1 0.05 49 ± 0 48 ± 1.15 0.25 63 ± 4.9 61 ± 9.16

TABLE 2 (Comparative example)

Concentration antibodies Percentage share of bonds (gg / ml) In the begining After 2 hours incubation 0.4 31 ± 0.64 0.76 ± 0.55 0.2 56 ± 1.6 6.7 ± 1.2 0.1 85 ± 0 30 ± 3.6

It can be seen from the results that the percentage of antibody binding does not change significantly after 2 hours for the immunoparticles of the present invention, while the proportion of these binding decreases considerably for PIHCA nanoparticles according to the control embodiment.

Example 3 --------. . .

Preparation of 1-ethoxycarbonyl-1-ethoxycarbonylmethyleneoxycarbonylethene immuno-nanoparticles coated with anti-CD4 0KT4-A antibodies and study of their stability.

The nanoparticles prepared according to step (1) in Example 1 were diluted to a concentration of 2.7 mg / ml using phosphate buffer at pH 7.4, and then 100 μΐ of 0KT4 monoclonal antibodies was added to 450 μΐ of this nanoparticle suspension. -A (50 µg / ml solution containing 0.1% bovine serum albumin; Ortho Diagnostic Systems, USA) labeled with fluorescein isothiocyanate. This mixture was then incubated at 20 ° C for 2 hours with stirring.

The stability of these immunoparticles was then tested using the same experimental conditions as in Example 2 above.

The results obtained in this test are shown in Table 3 below.

TABLE 3

Concentration antibodies (gg / ml) Percentage share of bonds In the begining After 2 hours incubation o, i - - 25- ± 5 14 £ 2 0.05 44 ± 3 30 ± 0 0.25 43 ± 6 30 ± 0

Example 4

Preparation of 1-ethoxycarbonyl-1-ethoxycarbonylmethyleneoxycarbonylethene immuno-nanoparticles coated with anti-CD4 0KT4 antibodies and study of their stability.

Immunoparticles were prepared as described in Example 3 using 100 μ fluores of fluorescein isothiocyanate labeled 0KT4 monoclonal antibodies (Ortho Diagnostic Systems, USA).

The stability of these immunoparticles was tested under the same experimental conditions as in Example 2.

The results obtained in this test are shown in Table 4 below.

TABLE 4

Concentration antibodies (gg / ml) Percentage of links In the beginning After 2 hours incubation 0.1 0.05 0.25 21 ± 0 16 ± 2 21 ± 5 21 ± 5 0 0

The above results show only insignificant changes in the percentage of antibodies bound after 2 hours of incubation.

Example 5

Study of the inhibitory potency of the immuno-particles of Example 1 with respect to syncytium formation using an in vitro model.

(1) Lymphocyte cultivation and HIV-1 virus infection.

Cell culture and HIV-1 virus infection were performed in the same manner as described by F. Rey et al., Virology, 1991, 181, 165-171.

CEM and MT4 cell lines were used to perform this assay. Infection with HIV-1 BRU virus (see:

F. Barré-Sinoussi et al., Science, 1983, 220, 868-871) was performed using a cell suspension containing 2. 10 ⁶ cells / ml, incubating at 37 ° C for 1 hour using a virus stock of equivalent volume. After infection, cells were washed by centrifugation with PBS (four times) to remove unbound (or free) virus particles.

These cells were then cultured at a concentration of 5 x 10 ³ cells / ml in RPMI 1640 medium (Gibco, USA) containing phenol red and addition of 10% fetal calf serum, 1% antibiotics and 1% glutamine. Every 3 or 4 days, these cells were diluted 3-fold (MT4 cells) or 4-fold (CEM cells) and then reclaimed in a new culture medium. Lymphocyte infection was demonstrated in a reverse transcriptase assay.

(2) Inhibition of syncytium formation.

As stated in F. Reye et al., Cited above, syncytia usually occurs after 1 or 2 days of incubation and after one volume of chronically infected CEM cells has been mixed with two volumes of 2 MT4 cells. 10 ⁵ cells / ml.

The ability of anti-CD4 monoclonal antibodies and immunoparticles bearing these anti-CD4 antibodies to inhibit syncytium formation was evaluated by incubating MT4 cells with various preparations for 2 hours, after which infected CEM cells were added.

Syncytium formation, as indicated by the + sign, was evaluated under a microscope 2 days after incubation at 37 ° C.

The results obtained in performing these tests are shown in Tables 5, 6 and 7 below, where:

- Table 5 shows the results obtained with iraunonanoparticles according to the invention carrying 10T4a antibodies.

- Table 6 of the comparative example shows the results obtained with PIHCA nanoparticles coated with 10T4a antibodies, and

Table 7, which also relates to the comparative example, shows the results obtained with unbound IOT4a antibodies.

TABLE 5

Inhibiting syncytium formation by the immuno-particles of the present invention.

Polymer (gg / ml) Antibodies total (gg / ml) Bound antibodies (gg / ml) Creation syncytia 25 0.05 0.012 - 25 0,025 0.012 - / + 25 0.012 0.0069 + 25 0.006 0.004 + 25 0 0 + 25 0 0 +

TABLE 6

Inhibition of syncytium formation by PIHCA nanoparticles carrying IO4a antibodies (comparative example)

Polymer (gg / ml) Antibodies total (gg / ml) Bound antibodies (µg / ml) Creation syncytia 5 0,045 0.012 + 5 0.02 0.011 + 5 0.01 0.008 + 5 0.005 0.005 +

Note: + presence of syncytium

- absence of syncytia

TABLE 7

Inhibition of syncytium formation by unbound IOT4a antibodies (comparative example)

Concentration Syncytia formation (gg / ml) io - 5 - 1 + 0.5 + 0.1 +

The results show that the use of the immuno-particles of the invention inhibits syncytium formation at an antibody concentration 100-fold less than that of the free (or unbound) antibody. In particular, complete inhibition of syncytium formation is achieved at an antibody concentration of 0.05 gg / mlr if 1-R is - the antibodies are in the form of immuno-particles according to the invention, and to achieve the same result with free antibodies (or unbound antibodies) concentrations of 5 gg / ml are necessary. In addition, these advantageous results have not been found with nanoparticles composed of polymers other than the immuno-nanoparticles of the present invention.

Example 6

Inhibitory studies of the immuno-nanoparticles of Example 3 with respect to syncytium formation in the in vit no model.

The immuno-particles of Example 3 were coated with anti-CD4 OKT4-A monoclonal antibodies and tested in the same manner as in Example 5.

The results obtained in this test are shown in Table 8 below.

TABLE 8

Polymer (gg / ml) Antibodies total (gg / ml) Bound antibodies (gg / ml) Creation syncytia 80 0.5 0,044 - 80 0.2 0,044 - / + 80 0.11 0,048 +

Example 7

Study of the inhibitory activity of the immunoparticles of Example 1 with respect to syncytium formation in an in vitro model using PAS 246 viral species.

Immunoparticles prepared as described in Example 1 and coated with anti-CD4 10T4a antibodies were tested in the same experimental procedure as described in Example 5, replacing HIV-1 BRU with PAS 246 (see F. Rey). et al., 1991, 181, 165-171).

The results obtained in this test are shown in Table 9 below.

TABLE 9

Polymer (g / ml) Antibodies total (g / ml) Bound antibodies (g / ml) Creation syncytia 25 0 025 0.012 - / * 25 0.05 0.012 - 25 0.1 0.012 -

The results show that the inhibition of syncytium formation begins at an antibody concentration of 0.025 gg / ml, which inhibition is complete at an antibody concentration of 0.05 µg / ml.

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Claims (4)

PATENT - Claims Whose Immunonoparticles, characterized in that they consist of polymer nanoparticles of a methylidene malonate compound of the general formula I: COOR, / h ₂ c = c (I) \ C00- _{(CH2) n} -C00R ₂ wherein: Rl and _R2, which may be identical or different, represent a linear or branched alkyl group having 1-5 carbon atoms, n is a number 1 to 5, wherein the nanoparticles are coated with anti-CD4 antibodies. 2. The immuno-nanoparticle of claim 1, wherein said methylidene malonate compound is a compound of formula I wherein R1 = R2 = ethyl and _n is 1. Immunonanocytes according to either of Claims 1 or 2, characterized in that the anti-CD4 monoclonal antibodies are selected from the group consisting of OKT4, OKT4-A and OTO4a antibodies. Immunonanocytes according to any one of claims 1 to 3, characterized in that the anti-CD4 monoclonal antibody is an 10T4a antibody.