Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
1999-11-18
2003-04-01
Zeman, Mary K. (Department: 1631)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S069100, C435S239000, C424S184100, C424S189100, C424S204100
Reexamination Certificate
active
06541210
ABSTRACT:
DESCRIPTION
The subject of the present invention is a process for the preparation of immunogens or diagnostic reagents that mimic an antigen or a pathogenic organism specific to a disease, even if this is uncharacterized or even unknown (thereby including auto-immune diseases whose etiology and/or pathogenesis is known or unknown). This process is based on the existence and availability of antibodies, both monoclonal or polyclonal, or of serum containing antibodies, which react specifically with the organism causing the infection.
Antibodies suitable for use in this process can be specific for any antigen of interest for which an immunogen or diagnostic reagent that mimes the antigen is sought. The antigen can be a protein or peptide whether synthetic, derived from a natural source, or produced recombinantly; carbohydrate; polysaccharide; glycoprotein; hormone; receptor; antibody; virus; substrate; metabolite; transition state analog; cofactor; drug; dye; nutrient; growth factor; cellular component; oncogene product; bacteria and their extracellular products; mammalian cells and extracts therefrom including tumor cells, virus infected cells and normal cells; parasites; protozoa; malarial antigens; helminths; fungi; rickettsia; or an allergen including but not limited to pollens, dusts, danders or extracts of the same; or a venom, poison, toxin, or toxoid; nucleic acids including DNA; or any other antigen without limitation. Antigens of viruses which are suitable for use in the present invention include antigens from the viruses including but not limited to polio virus, influenza virus, HIV, HTLV, papilloma virus, adeno virus, parainfluenza virus, measles virus, mumps virus, respiratory syncytial virus, shipping fever virus, Western and Eastern encephalomyelitis virus, Japanese B encephalomyelitis virus, Russian spring-summer encephalomyelitis virus, hog cholera virus, hepatitis virus, pox virus, rabies, virus, distemper virus, herpes virus, cytomegalo virus, foot and mouth disease virus, rhinovirus, Newcastle disease virus, vaccinia virus; and pseudorabies virus. The mime can be an immunogen, a vaccine, an inhibitor or activator, etc. without limitation.
As is known, all vaccines and diagnostic reagents currently on sale or undergoing clinical tests are conventionally obtained by means of processes based on the manipulation, modification and/or adaptation of pathogenic organisms or components thereof. These methods have given good results, but are not without problems. The greatest limitation associated with these methods is connected with the fact that they depend upon the availability of information and/or material directly deriving from pathogenic organisms or components thereof.
Previous attempts to overcome the above described limitation have so far failed for a lack of an efficient and reproducible experimental protocol; in particular, these attempts did not provide sufficient information in order to identify and characterize immunogenic mimics to be used for diagnosis and vaccine therapy. It must be emphasized that the present invention is focused on the development of a new technology aimed at overcoming conceptual and technical inadequacies of previously proposed protocols.
A key feature of the present invention is a novel strategy for the selection of antigenic and immunogenic mimics, based on the use, as reagents, of serum samples from patients and a counter-selection step utilizing serum samples from healthy individuals.
Use of the process for preparation according to the present invention allows this limitation to be overcome, furthermore offering additional advantages which will be clear from the following description.
The process for the preparation of immunogens or diagnostic reagents that mimic an antigen or a pathogenic organism specific to a disease—according to the present invention—is essentially characterized by the following operations:
identification of at least one antibody that reacts with the antigen or pathogenic organism specific to the disease;
construction of phage libraries which display on the surface of the capsid oligopeptides, expressed from random sequence oligonucleotidic inserts introduced into a gene coding for the phage capsid using genetic manipulation techniques;
selection of the phages that display on the surfaces of the capsid antigenic oligopeptides with a first pathologic serum in order to identify phage that display oligopeptides that react with all or most of the sera tested, and counter-screening with a panel of sera from another set of individuals taken as control in order to identify oligopeptides that do not react with all or most sera from controlled individuals;
optional use of the selected phages and/or fragments thereof and/or their derivatives for the formulation of diagnostic kits for the specific pathogenic agent, or in general for the diseases, including immunological disorders typical of so-called autoimmune diseases, with known or unknown etiology and/or pathogenesis;
optional use of the selected phages and/or fragments thereof and/or their derivatives for the formulation of an antagonist of the antigen-antibody reactions for treatment of the disease induced by said antigen;
optional use of the selected phages and/or fragments thereof and/or their derivatives to induce a tolerance of the phenomena of hypersensitivity and/or allergy to compounds and/or natural or synthetic preparations;
optional immunization of an organism by means of the selected phages and/or fragments thereof and/or their derivatives; and
optional verification of the presence, in the serum of the immunized organism, of antibodies that recognize the above antigen or organism specific to the disease.
The construction of phage libraries, according to the present invention, can be advantageously performed using the filamentous phages M13, F1 and Fd, or derivatives thereof. The reasons for this are the following:
filamentous phages are commonly used as molecular vectors in the field of molecular biology and genetic engineering. For example, by taking advantage of their feature to contain a genome with a single DNA helix, they have been particularly used in DNA sequencing experiments, in direct site mutagenesis experiments and for the expression of proteins and peptides;
the information required and sufficient for encapsidation of a single chain DNA genome has been well characterized and can be transferred to other molecular vectors;
it has likewise been demonstrated that at least two proteins of the capsid of filamentous phages can be modified by means of the addition or insertion of additional amino acid sequences. The resulting phages are encapsidated, maintain their ability to replicate and, in most cases, to infect bacterial cells. The foreign amino acid sequences are displayed on the surface of the phage, and can be recognized by interaction with antibodies or with other specific molecules according to the case.
The antibodies that can be used in the process for the preparation of immunogens and diagnostic reagents according to the present invention can be monoclonal antibodies, polyclonal antibodies, or antibodies contained in sera. The latter form of embodiment is of particular interest, because it provides for the first time a reproducible experimental strategy to identify novel antigenic and immunogenic mimics in absence of any information on the structure and properties of the natural and pathological antigen.
The gene coding for the phage capsid, with random sequence oligonucleotidic inserts, can be the gene coding for the protein VIII of the phage capsid or the gene coding for the protein III of said capsid.
The process according to the invention can be applied without restriction to any antibody or organism responsible for illness. Good results have been obtained using monoclonal antibodies, or sera specific for the surface antigen of the human hepatitis B virus (HBsAg).
The antigenic oligopeptides recognized by the antibodies used can be obtained by expression from random sequence oligonucleotidic inserts, using as a vector, for example
Cortese Riccardo
Felici Franco
Luzzago Alessandra
Monaci Paolo
Nicosia Alfredo
Browdy and Neimark , P.L.L.C.
Clow Lori A.
Istituto di Recerche di Biologia Moleculare P. Angeletti S.p.A.
Zeman Mary K.
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