Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
1999-11-29
2003-09-16
Wang, Andrew (Department: 1627)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S004000, C435S006120, C435S069100, C435S069600, C435S091500, C435S091500, C435S091500, C436S501000, C436S547000
Reexamination Certificate
active
06620587
ABSTRACT:
BACKGROUND TO THE INVENTION
A current focus of interest in molecular biology and biotechnology is in the display of large libraries of proteins and peptides and in means of searching them by affinity selection. The key to genetic exploitation of a selection method is a physical link between individual molecules of the library (phenotype) and the genetic information encoding them (genotype). A number of cell-based methods are available, such as on the surfaces of phages (1), bacteria (2) and animal viruses (3). Of these, the most widely used is phage display, in which proteins or peptides are expressed individually on the surface of phage as fusions to a coat protein, while the same phage particle carries the DNA encoding the protein or peptide. Selection of the phage is achieved through a specific binding reaction involving recognition of the protein or peptide, enabling the particular phage to be isolated and cloned and the DNA for the protein or peptide to be recovered and propagated or expressed.
A particularly desirable application of display technology is the selection of antibody combining sites from combinatorial libraries (4). Screening for high affinity antibodies to specific antigens has been widely carried out by phage display of antibody fragments (4). Combinations of the variable (V) regions of heavy (H) and light (L) chains are displayed on the phage surface and recombinant phage are selected by binding to immobilised antigen. Single-chain (sc) Fv fragments, in which the V
H
and V
L
domains are linked by a flexible linker peptide, have been widely used to construct such libraries. Another type of single chain antibody fragment is termed V
H
/K, in which the V
H
domain is linked to the complete light chain, i.e. V
H
-linker-V
L
-C
L
(10). This has a number of advantages, including stability of expression in
E. coli
and the use of the C
L
domain as a spacer and as a tag in detection systems such as ELISA and Western blotting. Antibody V
H
and V
L
region genes are readily obtained by PCR and can be recombined at random to produce large libraries of fragments (21). Such libraries may be obtained from normal or immune B lymphocytes of any mammalian species or constructed artificially from cloned gene fragments with synthetic H-CDR3 regions (third complementarity determining region of the heavy chain) generated in vitro (22). Single chain antibody libraries are potentially of a size of >10
10
members. Libraries can also be generated by mutagenesis of cloned DNA fragments encoding specific V
H
/V
L
combinations and screened for mutants having improved properties of affinity or specificity. Mutagenesis is carried out preferably on the CDR regions, and particularly on the highly variable H-CDR3, where the potential number of variants which could be constructed from a region of 10 amino acids is 20
10
or 10
13
.
It is clear that for efficient antibody display it is necessary to have a means of producing and selecting from very large libraries. However, the size of the libraries which can potentially be produced exceeds by several orders of magnitude the ability of current technologies to display all the members. Thus, the generation of phage display libraries requires bacterial transformation with DNA, but the low efficiency of DNA uptake by bacteria means that a typical number of transformants which can be obtained is only 10
7
-10
9
per transformation. While large phage display repertoires can be created (17), they require many repeated electroporations since transformation cannot be scaled up, making the process tedious or impractical. In addition to the limitations of transformation there are additional factors which reduce library diversity generated with bacteria, e.g. certain antibody fragments may not be secreted, may be proteolysed or form inclusion bodies, leading to the absence of such binding sites from the final library. These considerations apply to all cell-based methods. Thus for libraries with 10
10
or more members, only a small fraction of the potential library can be displayed and screened using current methodologies. As noted, the size of an antibody library generated either from animal or human B cells or artificially constructed can readily exceed 10
10
members, while the number of possible peptide sequences encoding a 10 residue sequence is 10
13
.
In order to avoid these limitations, alternative display systems have been sought, in particular in vitro methods which avoid the problem of transformation in library production. One such method is the display of proteins or peptides in nascent form on the surface of ribosomes, such that a stable complex with the encoding mRNA is also formed; the complexes are selected with a ligand for the protein or peptide and the genetic information obtained by reverse transcription of the isolated mRNA. This is known as ribosome or polysome display. A description of such a method is to be found in two U.S. patents, granted to G. Kawasaki/Optein Inc. (16). Therein, semi-random nucleotide sequences (as in a library) are attached to an ‘expression unit’ and transcribed in vitro; the resulting mRNAs are translated in vitro such that polysomes are produced; polysomes are selected by binding to a substance of interest and then disrupted; the released mRNA is recovered and used to construct cDNA. Two critical parts of the method are the stalling of the ribosome to produce stable complexes, for which cycloheximide is used, and the recovery of the mRNA, for which the bound polysomes are disrupted to release mRNA and the mRNA is then separately recovered. The latter is an integral part of the method as described by Kawasaki and adopted by all others until now. Thus, section VII of the patents (16) deals with the disruption of the polysomes by removal of magnesium, etc; no other method for recovery of RNA or cDNA is suggested other than ribosomal disruption. In U.S. Pat. No. 5,643,768, claim 1 refers to translating mRNA in such a way as to maintain polysomes with polypeptide chains attached, then contacting to a substance of interest, and finally isolating mRNA from the polysomes of interest. In claim 2, cDNA is constructed subsequent to isolating mRNA from the polysomes that specifically bind to the substance of interest. This is reiterated in claim 15, wherein step (g) comprises disrupting said polysomes to release said mRNA and step (h) comprises recovering said mRNA, thereby isolating a nucleotide sequence which encodes a polypeptide of interest. Similarly, this is repeated again in claim 29 (e) . . . isolating mRNA from the polysomes that specifically react with the substance of interest. In U.S. Pat. No. 5,658,754, claim 1 (g) also requires disrupting said polysomes to release mRNA; (h) is recovering said mRNA; and (i) is constructing cDNA from said recovered mRNA. However, Kawasaki did not reduce the method to practice in these filings and provided no results. Accordingly, the method was not optimised and he was unaware of the inefficiency of the system as he described it, in particular that due to the method of recovery of mRNA by polysome disruption.
Another description of prokaryotic polysome display, this time reduced to practice, is the international published application WO 95/11922 by Affymax Technologies (18) and the associated publication of Mattheakis et al. (14). Both relate to affinity screening of polysomes displaying nascent peptides, while the patent filing also claims screening of antibody libraries similarly displayed on polysomes. They refer to libraries of polysomes, specifically generated in the
E. coli
S30 system in which transcription and translation are coupled. To produce a population of stalled polysomes, agents such as rifampicin or chloramphenicol, which block prokaryotic translation, are added. The means of recovering the genetic information following selection of stalled ribosomes is again by elution of the mRNA. In the flowsheet of the method shown in
FIG. 10
of the patent application (18), an integral part is step 4, namely elution of mRNA from the ribosome complexes p
He Mingyue
Taussig Michael John
Discerna Limited
Friend Thomas
Heller Ehrman White & McAuliffe
Wang Andrew
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