Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing compound containing saccharide radical
Reexamination Certificate
1999-01-22
2001-01-16
Schwartzman, Robert A. (Department: 1636)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing compound containing saccharide radical
C435S477000, C435S488000
Reexamination Certificate
active
06174708
ABSTRACT:
Antibody molecules are constituted by a combination of two heavy chains (H) and two light chains (L) connected by disulphide bridges. The two heavy chains are joined together in a Y shaped structure and the two light chains are respectively bonded to the two branches of that structure, so that the variable regions of the light chains (V
L
) and heavy chains (V
H
) are located next to each other. Binding to an antigen results from the properties of the variable portions of the light and heavy chains. A complex rearrangement and selection system can rapidly induce a large quantity of antibodies specifically directed against an antigen.
The conventional hybridoma technique can be used to select clones of hybrid cells expressing genes coding for the light and heavy chains of an antibody molecule. This technique necessitates the fusion of cells of lymphocytic origin, containing the genes for antibody formation and cells forming immortal lines. The cells carrying the genes in question are generally obtained by random creation of libraries of circulating cells with or without prior immunisation with the specific antigen, and screening of the hybridomas by means of an antigen-antibody reaction after multiplication and culture of the hybridoma clones. This technique is difficult, the yield is limited, and screening is not easy.
A further method using recombinant bacteriophages has recently been used. The principles and the various implementations of that method have been described, for example, by D. R. Burton, Tiptech—May 1991, vol. 9, 169-175; D. J. Chiswell et al., Tiptech—March 1992, vol. 10, 80-84; H. R. Hoogenboom et al., Rev. Fr. Transfus. H{acute over (e)}mobiol., 1993, 36, 19-47 (see also International patent applications PCT WO 92/01047 and 92/20791).
That technique consists of inserting a repertoire of genes for variable antibody regions in association with a bacteriophage gene into a vector under conditions which enable expression of the gene in the form of a fusion protein at the phage surface, exposing the variable regions of the light and heavy chains bonded by their disulphide bridges in the manner of a Fab antibody fragment, and directly selecting the phages by a rapid separation method using the immobilised specific antigen, for example by immunoaffinity chromatography. After elution, the selected phages can infect a bacterium and be used for direct production or to repeat the selection cycles. That method is particularly powerful as in theory very large libraries can be created and screening of the library is very fine, efficient and rapid. One phage which is particularly suitable for that method is the filamentous phage fd, into which the fragment coding for one of the heavy and light chains of the antibody can be fused with the gene for the minor surface protein and into which the fragment coding for the other chain can be inserted, so that after infecting the bacteria with the phage, a population of phages is obtained carrying a fusion protein at their surface with the heavy and light chains in a configuration which is capable of recognizing the antigen, and is thus suitable for screening.
In addition to its simplicity, the advantages of that technique are enormous. Combined with prior amplification of the antibody gene library, a phage with a specific antibody fragment can be selected in a very large population of phages, in the order of 10
7
, which means that human antibody genes can be researched without being obliged to immunise the donor first.
Phages which randomly combine a light chain and a heavy chain can be obtained by cleaving followed by re-ligation or from two separate libraries of light chain genes and heavy chain genes.
However, the number of different clones which can be obtained is limited by the selection yield and by the degree of efficiency of the bacterial transformation.
One way of increasing the number of successful associations combining the light chains of a first library with the heavy chains of a second library has been described by P. WATERHOUSE et al., Nucleic Acids Research, 1993, vol. 21, No. 9, 2265-2266. Up to 10
12
clones can be obtained using a system of specific recombination of loxP sites sensitive to the action of Cre recombinase. However, the combination is reversible. Further, there is no control of the action of the recombinase and the recombinant vectors have no selective advantage over other vectors.
Considering the yield of the recombinant phage selection step which in reality retains only a fraction of the phages of interest, it would be desirable to obtain recombinant vector yields which were as high as possible with as few as possible non-recombinant vectors.
In a previous application (WO 95/21914 filed 2
nd
February 1995 with French priority FR 94 01519 of 10
th
February 1994), the inventors of the present invention described a method for the production of multicombinatorial libraries, in particular in the form of phages or phagemids, from two repertoires of genes, one for light chains and the other for heavy chains, to obtain a high number of clones.
That system has improved non-reversibility and selectivity properties since a new selection marker appears after recombination.
The non-reversibility properties are due to the absence of excision means in the vectors and in the host strain. The examples illustrating that application are thus characterised by the absence of the Xis excision protein in the vectors and in the Xis strain.
Further, the stable character of the joining sequences from recombination of specific recombination sites contributes to the non-reversibility of the system.
The present invention aims to improve that method further, in particular to increase its yield, the ease of implementing it and the diversity of the generated clones.
To this end, after two recombination events, the invention exchanges sequences between the two vectors. This exchange gives rise to two recombinant vectors, one with the two transcription units for the heavy and light chains but which is smaller in size than that of a vector which would result from fusion between the two starting vectors. As the final vector is smaller, its replication and packaging are more efficient and production is also improved, thus increasing the final number of clones.
The invention can also considerably broaden the choice of strains which can be used as host cells, in that it is no longer necessary for the strain used to possess the gene for integrase in its genome, that gene currently being carried by one of the two starting vectors and being found in the final vector.
This means that one is no longer restricted to strains with the gene for integrase integrated into their genome and any highly infectious strain producing phages can be selected (TG1, 71-18 or NM522).
Strains 71-18 (Stratagene; Yanisch-Perron, C. et al., (1985) Gene 33, 103-109) and NM522 (New England Biolabs; Woodcock, D. L., et al., (1989) Nucl. Acids Res. 17, 1563-1575) have proved to be particularly good phage producers. They can multiply the number of phages produced by a factor of 10 to 50 with respect to the D1210HP
E. coli
strain produced by Stratagene.
The invention provides a method for the production of multicombinatorial libraries in which, starting from a first repertoire of genes coding for a population of one of two types of polypeptides, which can covalently or otherwise combine, in particular variable regions of one of the antibody light or antibody heavy chains, and at least one gene coding for the other type of polypeptide, in particular a variable region of the other type, an antibody chain or preferably a second repertoire of genes coding for a population of said other type, introducing the genes of said first repertoire into a first starting vector to form a population of final vectors carrying different genes of said first repertoire, and introducing said gene of said other type or genes of said second repertoire into a second starting vector, and recombining said first and second starting vectors under conditions under which one of the vectors will
Aujame Luc
Bouchardon Annabelle
Geoffroy Fr{acute over (e)}d{acute over (e)}rique
Sodoyer Regis
McDonnell & Boehnen Hulbert & Berghoff
Pasteur Merieux Serums & Vaccins
Schwartzman Robert A.
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