Antigenic peptides

Details Antigenic peptides Antigenic peptides

1998-10-23

2001-04-24

Huff, Sheela (Department: 1642)

Chemistry: natural resins or derivatives; peptides or proteins;

Peptides of 3 to 100 amino acid residues

C530S331000, C530S324000, C530S350000

Reexamination Certificate

active

06222011

ABSTRACT:

This application is based on application No. MI95 A 002582 filed in Italy, the content of which is incorporated hereinto by reference.
This invention relates to improved antigenic peptides, methods for the preparation thereof, and use thereof.
It is known that antibodies recognise proteins important in diagnostic via binding to specific protein epitopes, formed generally by sequences 5 to 20 amino acids long.
In many cases, synthetic peptides corresponding to protein epitopes have been used for the preparation of diagnostic kits useful for the identification and/or the quantitative determination of antibodies associated to several pathologies. Identification of protein epitopes relevant for diagnostic use is carried out by producing, via chemical synthesis, peptides corresponding to different portions of the protein under examination, and evaluating the antigenic behavior thereof by means of a panel of sera of other biological fluids containing the corresponding antibody.
Very often, protein epitope structure is complex and their synthesis expensive.
Furthermore, chemical modification in the peptides identified as epitopes may lead, in an unpredictable way, to variations of the capability of antibody recognition (antigenicity), methadologies of broad applicability for improving, by making it more simple, the chemical structure of an antigenic peptide without altering the antigenic properties thereof are not yet available.
Therefore, there is still a great demand of an improvement in the structure of an antigenic peptide allowing manufacturing it at a lower cost, increasing the stability thereof against proteases, increasing the chemical stability thereof, and/or reducing aspecific interactions with other antibodies (antigenic cross-reactivity), without altering antigenic properties thereof.
Obviously, these advantages would have repercussions on the production of diagnostic kits, the use of said antigenic peptides as specific ligands for the purification of antibodies for diagnostic or therapy applications, and on the production of synthetic vaccines.
Surprisingly, it has now been found that these goals are reached when substituting one or more amino acids in the antigenic peptide sequence odd position, or respectively even, position with a glycine residue.
It is therefore a first object of this invention to provide an improved antigenic peptide, characterized in that the amino acid sequence, both normal and retro, is modified by replacing one or more amino acids with glycine, and that, when more than one amino acid is replaced, all the replaced amino acids are in odd position, or respectively, in even position, the amino acids having L or D configuration.
It is therefore a second object of this invention to provide a method for improving an antigenic peptide having a known amino sequence, characterized in that at least one amino acid in the sequence is replaced with glycine and that, when more than one amino acid is replaced, all the amino acids are replaced in the sequence even position, or respectively in the sequence odd position, and in that improved antigenic peptide, the amino acids having L or D configuration.
For sake of clarity, an antigenic peptide whose sequence is known and wherein, in the normal or retro sequence, at least one amino acid is replaced with glycine, will be hereinafter mentioned as “parent antigenic peptide”.
Many are the “parent antigenic peptides” used in diagnostics for the preparation of kits employed for the determination of specific antibodies in serum and/or other biological fluids. For example, for HIV 1 or 2 infection diagnosis, peptides corresponding to the 586-610 env region of HIV virus are used, while for hepatitis C (HCV) infection in diagnostic kits are used peptides corresponding to regions E1, NS4, and NS5. A list of Chemical Abstract Registry Numbers to antigenic peptides (i.e. “parent antigenic peptides”) which may be improved according to this invention is shown in the enclosed Table.
It is a third object of this invention to provide a diagnostic kit, characterized in that it contains at least one improved antigenic peptide as defined above.
It is a fourth object of this invention to provide the production of mono- or polyclonal antibodies able to recognize a respective “parent antigenic peptide”, characterized in that a suitable animal is immunized with a corresponding improved antigenic peptide of this invention, blood or other suitable tissues are taken after a suitable time and the desired antibody is isolated according to conventional techniques, such as chromatography and precipitation. As it is known to the persons skilled in the art, blood is taken for the preparation of polyclonal antibodies, while other tissues, such as the spleen, are taken for the preparation of polyclonal antibodies.
It is a fifth object of this invention to provide a vaccine, characterized in that it comprises at least one improved antigenic peptide as defined above.
It is a sixth object of this invention to provide a method for antibody purification according to the ligands technique, characterized in that the ligand is an improved antigenic peptide as defined above.
The selection of the aminoacids to be replaced in odd or in even position in the antigenic peptide sequence should be driven by criteria well known to the persons skilled in the art, such as the cost, the coupling yield with the preceding and following amino acids, the complexity of protection and deprotection of the reactive groups, the chemical and enzymatic stability, and the like.
A typical example of amino acids which may be preferably substituted with glycine comprise: cysteine, arginine, asparagine, aspartic acid, glutamic acid, glutamine, histidine, lysine, serine, tyrosine, threonine, and tryptophan.
The improved antigenic peptides according to this invention may be easily prepared according to conventional techniques of the peptide chemistry which comprise suitable protection of amino acids, coupling of protected amino acids and removal of protecting groups.
Preferably, they are prepared according to the solid phase synthesis technique, which comprises the following steps:
coupling , using suitable reactants, of the first amino acid from the C-terminus protected by suitable groups both in the amino group and, when required, in the side chain, on a suitable support for solid phase synthesis,
removal of the protecting groups at the amino group with suitable reactants,
coupling of the following amino acid from the C-terminus, protected at the amino group and, when required, in the side chain,
removal of the protecting group at the amino group and repetition of the coupling and deprotection cycles until all the amino acids comprised in the peptide sequence have been assembled,
removal of all remaining side chain protecting groups from the assembled antigenic peptide and cleavage from the resin.
These techniques are widely described in the literature and well known to the persons skilled in the art. See, for example, Atherton & Sheppard, 1989, Solid Phase Peptide Synthesis: A practical approach, IRL Press, Oxford, UK.
Generally, the diagnostic use of the compounds of this invention in procedures for antibody determination comprises the formation of complexes with antibodies raised against the “parent antigenic peptide”.
Typically, this technique comprises:
1) immobilizing at least one improved antigenic peptide of this invention on plastic ELISA microtiter plates, or on particles useful for diagnostic applications, either as they are or in form of conjugates with other molecules;
2) incubating a sample containing the desired antibody with the immobilized improved antigenic peptide;
3) washing the complex immobilized improved antigenic peptide/antibody; and
4) detecting the adsorbed antibody by means of a suitable reactant.
These techniques are widely described in the literature and are well known to the persons skilled in the art. See for example “Immunochemistry in Practice”, Johnstone & Thorpe, (1987) Blackwell, Oxford, UK.
Preferably, step 1 is carried out using plastic microt

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