Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
1998-03-16
2001-06-12
Saunders, David (Department: 1644)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C435S006120, C435S252300, C435S254200, C435S320100, C435S362000, C514S04400A, C514S885000
Reexamination Certificate
active
06245904
ABSTRACT:
FIELD OF THE INVENTION
The present invention is relative to nucleic acids containing at least one nucleic acid sequence coding for a recombinant polypeptide, which recombinant polypeptide contains at least partially the primary sequence of the invariant chain and at least one primary sequence from a specific T-cell epitope or from a protein and is processed in a desired manner in antigen-presenting cells for a binding of the specific T-cell epitope to MHC class II molecules.
No suitable vaccines are available against numerous pathogens. In the traditional methods of vaccination dead or attenuated pathogens are used as vaccine. In order to prevent undesired side effects or even the outbreak of an illness the attempt was made in individual instances to limit vaccines to a few defined antigens which can be produced by molecular, biologic methods in almost pure form and to nevertheless assure an efficient vaccine protection (e.g. hepatitis B vaccination with a recombinant HBsAg (“hepatitis B surface antigen”)).
BACKGROUND OF THE INVENTION
The vaccines previously known in the state of the art tend to bring forth side effects, among other things on account of the broadness of their immunogenicity. For example, a single viral or bacterial antigen usually consists of several hundred amino acids whereas T-cells recognize only a small section (<20 amino acids).
In order to utilize the specificity of T-cells, vaccines with a peptide base were developed, among other things. However, peptides have several disadvantages such as a short half-life, an unpredictable and non-specific distribution in the organism, low uptake into the cell and an unclear processing pathway. These factors obligatorily mean that from a physiological standpoint very high concentrations of peptides must be used in order to achieve a desired immune response. This naturally sharply limits the use of peptides as specific immunotherapeutic agents.
Antigens are presented as protein fragments on the surface of so-called “antigen-presenting cells” and are recognized by T-lymphocytes as effector cells. The MHC class I and class II molecules function thereby as peptide receptors in the antigen-presenting cells. In order to assure a functional dichotomy the MHC molecules are loaded in an exactly regulated manner in certain MHC loading compartments with the processed antigen peptides.
MHC molecules are representatives of a polymorphous gene family which is chromosomally coded in a special region, the major histocompatibility complex, “MHC”. The MHC molecules in humans are designated as HLA (human leucocyte antigen) molecules. MHC class I molecules are loaded directly at the site of biosynthesis with de novo translated virus or tumor antigens but also with endogenous, cytosolic protein fragments. In contrast thereto, MHC class II molecules (designated in the following in brief as “MHC II molecules”) are charged in cellular compartments which communicate with the extracellular environment. In humans the MHC II molecules comprise the HLA-DR, HLA-DQ and HLA-DP molecules, which occur in various genetically coded alleles. Thus, e.g. bacterial antigens from the extracellular environment can be taken up and be presented after intracellular processing in the antigen-presenting cells on their cell surface.
In order to assure an effective immunosurveillance the physiology of the MHC molecules is designed in such a manner that they can present as broad a spectrum of antigenic peptides as possible. Consequently, the copy number of a defined antigenic peptide on the cell surface of antigen-presenting cells is very low (magnitude 10
2
of a defined antigenic peptide given a total population of approximately 10
5
peptide receptors). This means that a very heterogenous mixture consisting of a plurality of various antigenic peptides bound to MHC molecules (“peptide ligands”) is exposed on the cell surface of the antigen-presenting cells.
Experimental attempts to produce defined MHC/peptide complexes were based up to the present on the expression of recombinant MHC molecules e.g. in the Baculovirus- or
E. coli
expression system. These MHC II molecules have an empty binding groove and can be charged in vitro with suitable peptides. The binding/loading efficiency can vary greatly and can be used only for in vitro tests.
Another attempt starts with recombinant MHC II molecules which, constructed as fusion protein (of the &bgr; chain), terminally exhibit a desired ligand via a flexible linker. This ligand segment spontaneously fills the binding groove, so that a more or less large number of MHC II molecules carries this defined peptide ligand as a function of the cell type used. This attempt is limited to the DNA transfection of host cells whereas a direct application on the protein level is not possible.
SUMMARY OF THE INVENTION
Thus, the present invention has the problem of reinforcing the antigenicity of proteins and/or of reducing the (intrinsic) heterogeneity of MHC II antigenic peptide complexes of antigen-presenting cells while avoiding, among other things, the above-mentioned disadvantages of the vaccination methods described in the state of the art. In particular, a novel system for a directed and highly specific stimulation of the immune system by means of exactly definable antigenic peptides should be made available in order e.g. to improve the vaccinations of mammals.
This problem is solved by the embodiments of the present invention characterized in the claims. In particular, a nucleic acid is made available in a first embodiment in accordance with the invention which contains at least one nucleic acid sequence coding for a recombinant polypeptide, which recombinant polypeptide contains at least partially the primary sequence of the invariant chain and at least one primary sequence from a specific T-cell epitope which is inserted into the area of the invariant chain comprising the CLIP sequence and which recombinant polypeptide is processed in such a manner in antigen-presenting cells that the specific T-cell epitope is produced and binds to MHC II molecules.
In a further embodiment of the invention a nucleic acid is made available which contains at least one nucleic acid sequence coding for a recombinant polypeptide, which recombinant polypeptide contains at least partially the primary sequence of the invariant chain and at least one primary sequence from a specific T-cell epitope, with at least one amino acid of the area of the invariant chain comprising the CLIP sequence being deleted and replaced by the T-cell epitope, and which recombinant polypeptide is processed in such a manner in antigen-presenting cells that the specific T-cell epitope is produced and bonds to MHC II molecules.
REFERENCES:
Sanderson et al. PNAS 92:7217-7221, Aug. 1995.*
Tisch R & McDevitt, M.O. PNAS 91-437-438, Jan. 1994.*
Physicians Desk Reference 53rd Edition Foreword Arky Ronald Medical Economics Company Inc Montvale NJ USA, 1999.*
Merriam-Webster's Collegiate Dictionary 10th Edition p. 870 Merriam-Webster Inc USA, 1993.
Malcherek Georg
Melms Arthur
DeCloux Amy
Hardaway/Mann IP Group
Saunders David
The University of Tubingen
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