Expression screening vector

Details Expression screening vector Expression screening vector

1996-07-12

2001-04-10

Venkat, J. (Department: 1627)

Chemistry: molecular biology and microbiology

Vector, per se

C435S005000, C435S007100, C435S006120, C435S252300, C530S387100, C530S387300, C536S023100, C536S023400, C536S023500, C536S023530

Reexamination Certificate

active

06214613

ABSTRACT:

This application is a 317 of PCT/JP94/02033, filed on Dec. 2, 1994.
BACKGROUND OF THE INVENTION
1. Field of the Invention
This invention relates to a vector which directs expression of an antibody or a portion of antibody at least containing an antigen binding site (hereinafter designated as antibodies) on the surface of cell membrane, in order to select a nucleotide sequence coding for a variable region of the antibody for a specific antigen (hereinafter designated as nucleotide sequence of variable region), a library and a method for selecting a nucleotide sequence of antigen-specific antibody variable region using the said vector, and a kit containing the same.
2. Description of the Related Art
Development of antibody for application in pharmaceuticals has recently progressed. Antibody of those pharmaceutical applications is preferably a human type due to antigenicity to the humans. However, preparation of human antibody to the specific antigen has not been established unlike mouse monoclonal antibody with established hybridoma method. From these points of view, a development for a new process applicable to the preparation of human monoclonal antibody in place of hybridoma method, has been expected.
Preparation of specific antibody for specific antigen by means of genetic engineering process, requires a nucleotide sequence of variable regions of the H- and L-chains which determine the antigen specificity of the antibody. Genetically engineered preparation of antibody using the variable region of the H- and L-chains has been known [Xiang, J. et al. (1990), Mol. Immun., 27, 809; Bebbington, C. R. et al. (1992), Biotechnology, 10, 169].
A method for preparing the nucleotide sequence of variable region of antigen-specific H- and L-chains, using
E. coli
phage or phagemid has been known [Huse. W. D. et al. (1989). Science, 246, 1275; McCafferty, J. et al. (1990), Nature, 348, 552 and Kang, A. S. et al. (1991), Proc. Natl. Acad. Sci. USA 88, 44363].
In these methods, antibody library is prepared by producing Fab (refer to
FIG. 1
) per se, or antibody library (phage antibody library) is prepared by producing fused phage coat protein with Fab or a single chain Fv, which is prepared by linking the H-chain variable region to the L-chain variable region of antibody with suitable linker (refer to
FIG. 1
, hereinafter designates as scFv), thus antigen-specific antibody and gene thereof are selected by means of binding affinity with antigen.
As explained hereinabove, phage antibody library is prepared by producing Fab or scFv by
E. coli
. Fab or scFv produced by
E. coli
form an inclusion body (in case of intracellular production), or is accumulated in the periplasm to form insoluble protein (in case of secretion) [Anand, N. N. et al. (1991), J. Biol. Chem., 266, 21874; Huston, J. S. et al. (1991), Methods Enzymol., 203, 46]. Inclusion body and insoluble protein should be solubilized to construct higher-order structure (renature) for obtaining protein with antigen-binding activity.
Preparation of scFv by linking the H-chain variable region to L-chain variable region with suitable linker in various antibodies, for which properties thereof were well known, using expression system of
E. coli
has been tried [Bird, R. E. et al. (1991), TIBTEC, 9, 132; Anand, N. N. et al. (1991), J. Biol. Chem., 266, 21874]. Affinity of the scFv is generally decreased as compared with that of Fab obtained by papain hydrolysis. Further productivity of scFv varies greatly by an order of the H- and L-chain variable regions [Tsumoto, K. et al. (1994), Biochem. Biophys. Res. Comm., 201, 546]. Therefore production of scFv of some antibodies by
E. coli
may show disadvantages. In the phage antibody system hereinbefore explained, the scFv is prepared to form fused protein with minor coat protein g3p of fd phage. Fused protein of some kind of scFv loses antigen-binding activity [Furuta, S. et al., a presentation at Japan. Biochem. Soc. Meeting, 1994].
As explained hereinabove, the expression of scFv or phage antibody using scFv in
E. coli
has various disadvantages and the case with mere success in preparation of an antibody does not warrant the success in all other cases of the antibodies. There are possibilities to be prepared incomplete library having problems such that the expressed variable region has a bias in variety and that affinity of the expressed antibody differs from that of the original.
A problem in expression of scFv in
E. coli
seems to be caused by artificial structure linking the H-chain variable region to the L-chain variable region with linker and, in addition to that, is thought to be mainly caused by cellular structure of
E. coli
which has no organella for constructing higher-order structure of protein such as endoplasmic reticulum in animal cells. Originally, since the active antigen-binding site of antibody can be formed by establishing exact steric structure containing intramolecular disulfide bonds in H-chain and L-chain, respectively, and by forming the one to one corresponding dusulfide bond of the H- and L-chains,the higher-order structure of protein is extremely important for the expression of activity.
Expression system for Fab on the surface of phage in place of scFv has the same problem. Considering the large size of molecular weight of Fab of about 45,000 as compared with that of scFv of about 26,000, and necessity of exact association with two polypeptide chains, an expression of Fab having exact activity will be more difficult than that of scFv. Expression of active Fab in
E. coli
shows less productivity [Skerra, A. et al. (1991) Protein Eng. 4, 971].
As explained in the above, a screening system in use of
E. coli
is not preferable for expression of protein having complex higher-order structure such as antibody which is originally produced in animal cells. Accordingly, the expression screening system in use of eukaryotic cells, especially animal cells, is thought to be preferable. Expression system in use of animal host cells shows no problems as like in the expression system in
E. coli
[Wood, C. R. et al., (1990) J. Immunol., 145, 3011]. A library which expresses exact antigen-binding activity of the original antibody could be prepared, and the nucleotide sequence of variable region of antigen specific antibody could be screened effectively from the library. Natural human antibody is preferable for antibody therapeutics in view of antigenicity in human. A process for production of human antibody by means of recombinant DNA technology in use of animal host cells is preferable.
On the point that the final expression is performed by animal cells, use of animal cells at the screening step may be preferable, in order not to make difference in antigen-binding activity of antibody in the screening steps and the production steps.
As clearly explained hereinabove, although the screening system in
E. coli
for nucleotide sequence of variable region of antigen-specific H- and L-chain is known, development on more preferable screening system in use of eukaryotic cells, specifically animal cells is expected.
SUMMARY OF THE INVENTION
The present invention relates to a method for screening nucleotide sequence of variable region of antigen-specific antibody in use of eukaryotic cells, specifically animal cells, which are preferable for expression of antibody, and a vector therefor.
The inventors of the present invention have tried to find out the idea comprising preparing a vector which expresses antibodies on cell membrane, inserting nucleotide sequence of variable region of various antibody to the vector to express the antibodies on the cell membrane, concentrating the cells which express the antibodies binding to antigen in use of marker of antigen-binding activity, and obtaining the nucleotide sequence of variable region of antigen-specific antibody.
The present invention consists of the following concepts.
(1) An expression vector for preparing a library of an antibody variable region, which ca

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