United States Patent:  6,004,763

Assignee:  Institut Pasteur (FR)

Filed:  May 12, 1998

The invention concerns the use, in the induction of an immune response, of a synthetic microparticle polymer carrying on the surface one or more covalently bonded proteins capable of carrying one or more epitopes, the molecular weight of the protein(s) on the surface of the microparticles, being adjusted so as to direct the immune response to the induction of a humoral and cellular response or to the induction of a mainly cellular response, said microparticles have an average diameter of approximately 0.25 to 1.5 .mu.m.

SUMMARY OF THE INVENTION

The invention offers the development of products giving a good immune response with either a cellular or a humoral direction.

According to the invention, it has been found that such a response can be induced by using microparticles, of small size and having varied antigenic molecular weights.

The present invention particularly relates to synthetic polymer microparticles carrying on their surface at least one covalently bonded proteins, each carrying at least one epitope to induce an humoral or cellular response, the molecular weights of the proteins being adjusted to direct the said immune response towards the induction of cellular or humoral response.

In a preferred embodiment, the molecular weight of the protein is greater than 30 kD, and preferably greater than 50 kD, in which case the immune response is mainly a cellular response.

In another preferred embodiment, the molecular weight of the protein is lower than 30 kD, preferably lower than 15 kD, and the protein comprises B and T epitopes. The immune response is an humoral and cellular response.

The invention also relates to the characteristics below, considered alone or in all technically possible combinations.

The microparticles advantageously have an average diameter of between about 0.25 .mu.m and 1.5 .mu.m, and preferentially of about 1 .mu.m so as to be able to be presented to CD4⁺ T lymphocytes by phagocytic cells but not by B lymphocytes.

The coupling of the antigenic proteins or microparticles must be covalent in order to avoid the liberation of the antigen in soluble form.

Said microparticles are more particularly characterized in that the covalent bond is formed by reaction between the NH₂ and/or CO groups of the proteins and the material making up the microparticle.

Advantageously such bond is created by using a bridging reagent as intermediate, such as for example glutaraldehyde or carbodiimide. However, any other bifunctional reagent able to form such a bond can be used. Such reagents are known, see for example < >. This bond can also be formed without a bridging reagent.

The material of the microparticle can advantageously be a biocompatible polymer, such as an acrylic polymer, for example polyacrolein or polystyrene or the poly(alpha-hydroxy acids), copolymers of lactic and glycolic acids, or lactic acid polymers.

By polymer should be understood any homopolymer or hetero or copolymer.

It must allow covalent bonding of the proteins to the material and must not cause a rejection or toxic reaction by the organism into which it may be injected. Advantageously, for human therapeutic applications, it should be a biodegradable polymer, for example a polymer able to be degraded by cells containing lysosomal enzymes, such as the macrophages.

Such biodegradable materials can include lactic and glutamic acid polymers, starch or polymers used for biomedical applications, and in particular those used-for sutures.

Such microparticles can carry on their surface, in addition to the antigenic proteins, molecules able to activate the immune system, such as the interleukins, in particular gamma-interferon or interleukin 4.

The microparticles which are the object of the present invention can in addition be encapsulated in order to protect the antigens fixed to their surfaces from degradation and to transport them to their site of action.

They can thus comprise a nucleus formed from a polysaccharide matrix, to which are bound the antigens, an initial lipid layer bound covalently to the nucleus and a second layer of amphophilic molecules.

Another object of the invention is drugs or vaccines comprising the microparticles described above, as well as pharmaceutical compositions characterized in that they contain them, in combination with pharmaceutically compatible diluents or adjuvants.

The present invention relates furthermore to a method of inducing an immune response in warm blooded animals comprising administering to these animals an inducing amount of these microparticles.

These microparticles can carry one or more proteins which can themselves each contain one or more epitopes. Such proteins can be glycoproteins, synthetic peptides containing an epitope or several epitopes, or any other nonprotein molecule or molecule containing a protein portion able to induce an immune response.

The proteins and antigens covalently bonded to the microparticles depend on the anticipated application for said microparticles.

They also depend on the type of immune response required, but also on the disease or ailment to be treated or against which the patient is to be protected.

Examples of epitopes which may be used are the epitopes from the Pre S2 region of the HBS antigen of the viral hepatitis virus, with the following sequences:

T epitope: Pre S:T (120-132)

MQWNSTTFHQTLQ (SEQ ID NO:1)

B epitope: Pre S:B (132-145)

QDPRVRGLYFPAGG (SEQ ID NO:2)

Other examples are the epitopes of the VP1 protein of the poliomyelitis virus whose sequences are as follows:

T epitope: C3: T (103-115)

KLFAVWKITYKDT (SEQ ID NO:3)

B epitope: C3: B (93-103)

DNPASTTNKDK (SEQ ID NO:4)

Another example is the epitope of the V3 loop of the GP120 protein of the HIV1 virus whose sequence is the following:

T+B epitope: V3 loop

INCTRPNNNTRKSIRIQRGPGRAFVTIGKIGNMRQAHCNI (SEQ ID NO:5)

Such microparticles may be injected into patients who are to be treated in a therapeutic or prophylactic manner in ways known to those skilled in the art, for example by subcutaneous, intra-peritoneal, or intravenous injection or by any other means for inducing an immune response.

This subject is discussed in Current protocols in immunology (edited by J. P. Coligen, A. M. Kruisbeek, D. H. Margulies, E. M. Shevach, W. Strober, published by Wiley-Interscience), in which the range of immunological techniques is listed.

A particular advantage of the present invention rests in the fact that it enables the induction of a humoral or cellular immune response without the addition of adjuvants to the beads or microparticles. However, the addition of non toxic adjuvants not causing an immune side-reaction is also envisageable within the scope of use according to the present invention.

Claim 1 of 23 Claims

1. A method of inducing an immune response in warm-blooded animals comprising administering to warm-blooded animals an immune response inducing amount of synthetic biocompatible microparticles carrying on their surface at least one covalently bonded protein, each carrying at least one epitope to induce an humoral or cellular immune response and having an average diameter of between 0.25 .mu.m and 1.5 .mu.m, the molecular weight(s) of the protein(s) on the microparticle surfaces being adjusted to direct the said immune response towards the induction of cellular and/or humoral response.

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