Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving virus or bacteriophage
Patent
1996-10-07
1998-01-27
Mosher, Mary E.
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving virus or bacteriophage
435 71, 435 693, 435 696, 4351723, 435 697, 4352523, 43525233, C12Q 107, C12Q 102, G01N 3353, C12P 2104
Patent
active
057120897
DESCRIPTION:
BRIEF SUMMARY
TECHNICAL AREA OF THE INVENTION
The present invention concerns a method for selecting a molecule, such as an antibody, antigen, peptide, protein or fragment thereof, which molecule is expressed together with a phage coat protein on the phage's surface.
BACKGROUND OF THE INVENTION
Monoclonal antibodies were introduced in 1975 by George Kohler and Cesar Milstein. The concept comprises fusing immune B lymphocytes from mice with a tumour cell line, for instance a myeloma/plasmacytoma. The resulting hybrid myeloma (=hybridoma) will posses the following two distinct properties: 1. produce specific antibodies; and 2. live infinitely in cell culture. The first of these properties is inherited from the immune mouse cells, whereas the second one comes from the tumour cell line. The hybridoma prepared as outlined above, will produce so-called monoclonal antibodies of high specificity and in infinite amounts; properties which makes them especially suitable for use in biomedical applications.
Human therapy using monoclonal antibodies does however require human antibodies, among other because an unwanted glycosylation appears on the mouse antibodies, which renders these antibodies directly unsuitable for human therapy (Borrebaeck et al., 1993). Human monoclonal antibodies have however shown themselves to be considerably much harder to produce than the mouse antibodies, especially because human beings can not be immunised due to ethical considerations. This means that the starting material, i.e. the immune B lymphocytes, has not been optimal. The main problem has been that the number of immune B lymphocytes has been very low in non-immunised individuals, which makes it extremely difficult to select specific antibodies from said B lymphocytes.
In 1985 Smith (Smith, 1985) published a method which dramatically changed how antibodies and especially human antibodies could be produced. Smith showed how small peptides could be expressed together with a phage coat protein on a filamentous phage (virus which infects bacteria). As filamentous phages allow even foreign proteins to be expressed on some of their own coat proteins, such as for instance protein 3 or protein 8, these phages are very well suited for expression of even the relatively big antibody fragments, such as for instance Fab of Fv (McCaffery et al., 1990; Barbas et al., 1991; Huse 1991).
The method for placing the antibody fragment on the phage surface is the following:
From a starting material which comprises B lymphocytes, such as blood, lymphoid tissue or the like, the B lymphocytes are separated and a gene library of the antibodies produced by said B lymphocytes is erected. The genes encoding the variable heavy and light antibody domains (V.sub.H and V.sub.L) are amplified through the so-called PCR-method (PCR=Polymerase Chain Reaction), which was first described applied on antibodies by Larrick et al. (1989). These amplified gene segments, which codes for all different antibody specificities found in the starting material used, are thereafter cloned into a so-called phagemid vector with a random combination of different V.sub.H /V.sub.L genes (Huse et al., 1989). The result of this cloning is that all available specificities can be immortalised in one single step and in a following step they may be expressed on the surface of a filamentous phage together with for example coat protein 3. Those phages which express an antibody fragment with the sought after specificity can then be selected by taking advantage of the surface displayed antigen receptor, i.e. the antibody fragment. In summary, it can be said that all antibody specificities in a certain starting material can be directly immortalised by PCR amplification and thereafter expressed on the surface of a phage.
Theoretically this method gives access to the complete pool of antibodies found in the immune system. This pool consists of up to 10.sup.14 different antibody specificities and at a given point of time in a human beings life approximately 10.sup.8 -10.sup.9 different specificities will be availa
REFERENCES:
McCafferty et al., 1990, Nature, vol. 348; pp. 552-554.
Methods In Enzymology, vol. 217, 1993, G.P. Smith, et al "Libraries of Peptides and Proteins Displayed on Filamantous Phage" pp. 228-257.
Nucleic Acis Research, vol. 21, No. 9, 1993, P. Waterhouse, et al, "Combinatorial infection and in vivo recombination, a stragegy for making large phage antibody repertoires" pp. 2265-2266.
Proc. Natl. Acad. Sci., vol. 87, Apr. 1990, D.M. Kurnit, et al, "Improved genetic selection for screening bacteriophage libraries by homologous recombination in vivo" pp. 3166-3169.
Gene, vol. 109, 1991, W. Markland, et al, "Design, construction and function of a multicopy display vector using fusions to the major coat protein of bacteriophage M13" pp. 13-19.
Bio/Technology, vol. 12, Oct. 1994, M. Duenas et al., "Clonal Selection and Amplication of Phage Displayed Antibodies by Linking Antigen Recognition and Phage Replication" pp. 999-1002.
Borrebaeck Carl A. K.
Duenas Marta
BioInvent International AB
Mosher Mary E.
Salimi Ali R.
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