Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1992-09-04
2001-09-18
Ketter, James (Department: 1636)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S235100, C435S320100, C435S069600, C435S252330, C435S489000
Reexamination Certificate
active
06291161
ABSTRACT:
TECHNICAL FIELD
The present invention relates to a method for isolating a gene coding for a receptor having a preselected activity.
BACKGROUND
The immune system of a mammal is one of the most versatile biological systems as probably greater than 1.0×10
7
antibody specificities can be produced. Indeed, much of contemporary biological and medical research is directed toward tapping this repertoire. During the last decade there has been a dramatic increase in the ability to harness the output of the vast immunological repertoire. The development of the hybridoma methodology by Kohler and Milstein has made it possible to produce monoclonal antibodies, i.e., a composition of antibody molecules of a single specificity, from the repertoire of antibodies induced during an immune response.
Unfortunately, current methods for generating monoclonal antibodies are not capable of efficiently surveying the entire antibody response induced by a particular immunogen. In an individual animal there are at least 5-10,000 different B-cell clones capable of generating unique antibodies to a small relatively rigid immunogens, such as, for example dinitrophenol. Further, because of the process of somatic mutation during the generation of antibody diversity, essentially an unlimited number of unique antibody molecules may be generated. In contrast to this vast potential for different antibodies, current hybridoma methodologies typically yield only a few hundred different monoclonal antibodies per fusion.
Other difficulties in producing monoclonal antibodies with the hybridoma methodology include genetic instability and low production capacity of hybridoma cultures. One means by which the art has attempted to overcome these latter two problems has been to clone the immunoglobulin-producing genes from a particular hybridoma of interest into a procaryotic expression system. See, for example, Robinson et al., PCT Publication No. WO 89/0099; Winter et al., European Patent Publication No. 0239400; Reading, U.S. Pat. No. 4,714,681; and Cabilly et al., European Patent Publication No. 0125023.
The immunologic repertoire of vertebrates has recently been found to contain genes coding for immunoglobulins having catalytic activity. Tramontano et al.,
Sci.,
234:1566-1570 (1986); Pollack et al.,
Sci.,
234:1570-1573 (1986); Janda et al.,
Sci.,
241:1188-1191 (1988); and Janda et al.,
Sci.,
244:437-440 (1989). The presence of, or the ability to induce the repertoire to produce, antibodies molecules capable of a catalyzing chemical reaction, i.e., acting like enzymes, had previously been postulated almost 20 years ago by W. P. Jencks in
Catalysis in Chemistry and Enzymology,
McGraw-Hill, N.Y. (1969).
It is believed that one reason the art failed to isolate catalytic antibodies from the immunological repertoire earlier, and its failure to isolate many to date even after their actual discovery, is the inability to screen a large portion of the repertoire for the desired activity. Another reason is believed to be the bias of currently available screening techniques, such as the hybridoma technique, towards the production high affinity antibodies inherently designed for participation in the process of neutralization, as opposed to catalysis.
BRIEF SUMMARY OF THE INVENTION
The present invention provides a novel method for screening a larger portion of the immunological repertoire for receptors having a preselected activity than has heretofore been possible, thereby overcoming the before-mentioned inadequacies of the hybridoma technique.
In one embodiment, an immunoglobulin heavy chain variable region V
H
gene library containing a substantial portion of the V
H
gene repertoire of a vertebrate is synthesized. In preferred embodiments, the V
H
-coding gene library contains at least about 10
3
, preferably at least about 10
4
and more preferably at least about 10
5
different V
H
-coding nucleic acid strands referred to herein as V
H
-coding DNA homologs.
The gene library can be synthesized by either of two methods, depending on the starting material.
Where the starting material is a plurality of V
H
-coding genes, the repertoire is subjected to two distinct primer extension reactions. The first primer extension reaction uses a first polynucleotide synthesis primer capable of initiating the first reaction by hybridizing to a nucleotide sequence conserved (shared by a plurality of genes) within the repertoire. The first primer extension reaction produces a plurality of different V
H
-coding homolog complements (nucleic acid strands complementary to the genes in the repertoire).
The second primer extension reaction produces, using the complements as templates, a plurality of different V
H
-coding DNA homologs. The second primer extension reaction uses a second polynucleotide synthesis primer that is capable of initiating the second reaction by hybridizing to a nucleotide sequence conserved among a plurality of V
H
-coding gene complements.
Where the starting material is a plurality of complements of different V
H
-coding genes provided by a method other than the first primer extension reaction, the repertoire is subjected to the above- discussed second primer extension reaction. That is, where the starting material is a plurality of different V
H
-coding gene complements produced by a method such as denaturation of double strand genomic DNA, chemical synthesis and the like, the complements are subjected to a primer extension reaction using a polynucleotide synthesis primer that hybridizes to a plurality of the different V
H
-coding gene complements provided. Of course, if both a repertoire of V
H
-coding genes and their complements are present in the starting material, both approaches can be used in combination.
A V
H
-coding DNA homolog, i.e., a gene coding for a receptor capable of binding the preselected ligand, is then segregated from the library to produce the isolated gene. This is typically accomplished by operatively linking for expression a plurality of the different V
H
-coding DNA homologs of the library to an expression vector. The V
H
-expression vectors so produced are introduced into a population of compatible host cells, i.e., cells capable of expressing a gene operatively linked for expression to the vector. The transformants are cultured under conditions for expressing the receptor coded for by the V
H
-coding DNA homolog. The transformants are cloned and the clones are screened for expression of a receptor that binds the preselected ligand. Any of the suitable methods well known in the art for detecting the binding of a ligand to a receptor can be used. A transformant expressing the desired activity is then segregated from the population to produce the isolated gene.
In another embodiment, the present invention contemplates a gene library comprising an isolated admixture of at least about 10
3
, preferably at least about 10
4
and more preferably at least 10
5
V
H
- and/or V
L
-coding DNA homologs, a plurality of which share a conserved antigenic determinant. Preferably, the homologs are present in a medium suitable for in vitro manipulation, such as water, phosphate buffered saline and the like, which maintains the biological activity of the homologs.
A receptor having a preselected activity, preferably catalytic activity, produced by a method of the present invention, preferably a V
H
or F
V
as described herein, is also contemplated.
The present invention further contemplates a gene library comprising at least 10
5
different V
H
- or V
L
-coding DNA homologs. Each of the homologs is present in the library as a population of DNA strands of a first length to the number of the strands having a length other than the first length is at least 4:1.
REFERENCES:
patent: 4356270 (1982-10-01), Itakura
patent: 4642334 (1987-02-01), Moore et al.
patent: 4656134 (1987-04-01), Ringold
patent: 4683195 (1987-07-01), Mullis et al.
patent: 4683202 (1987-07-01), Mullis
patent: 4704692 (1987-11-01), Landner et al.
patent: 4711845 (1987-12-01), Gelfand et al.
patent: 4714681 (1987-12-01), Reading
patent:
Lerner Richard A.
Riechmann Lutz
Sorge Joseph A.
Winter Gregory P.
Halluin Albert P.
Howrey Simon Arnold & White , LLP
Ketter James
Kung Viola T.
Scripps Research Institute
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