Chemistry: molecular biology and microbiology – Virus or bacteriophage – except for viral vector or...
Reexamination Certificate
1999-01-21
2001-01-23
Patterson, Jr., Charles L. (Department: 1652)
Chemistry: molecular biology and microbiology
Virus or bacteriophage, except for viral vector or...
C435S188500
Reexamination Certificate
active
06177270
ABSTRACT:
FIELD OF THE INVENTION
This invention broadly relates to methods for selection for catalytic moieties, to methods for increasing the concentration of catalytic moieties in a sample containing catalytic moieties, and to substantially pure or concentrated catalytic products from such methods. This invention also relates to methods for selection or concentrating a population of recombinant viruses suspected of including viruses expressing catalytic antibodies or catalytic portions thereof. This invention also relates to detecting recombinant viruses that express a catalytic antibody or catalytic portions thereof and act catalytically. This invention further relates to a method for producing a recombinant virus or a cell-line expressing a catalytic antibody or catalytic portions thereof by infecting a host susceptible to infection by a recombinant virus that expresses a catalytic antibody or catalytic portion thereof. This invention also relates to substantially pure recombinant virus or cell populations which express a catalytic antibody or catalytic portion thereof from the aforesaid methods.
BACKGROUND OF THE INVENTION
Throughout this disclosure reference will be made to the published literature by numerals in parenthesis. These numerical references correspond to a listing of such literature references appearing at the end of this disclosure; all of these literature references being hereby incorporated herein by reference.
Viruses infect cells and divert the biosynthetic apparatus of the cell to synthesizing virus progeny. Certain viruses infect the host cell, cause the host DNA to break down and virus progeny to form in the cell whereupon the cell lyses with the release of mature virus progeny. Other viruses are lysogenic. These viruses infect a host cell and the viral DNA becomes inserted into a region of the host chromosome whereupon for generations the resultant cell-line (from replication of the infected cell) expresses genetic products of the virus. A progeny of the original infected cell can spontaneously release the viral DNA from its chromosome or be induced to do the same, whereupon a lytic cycle resulting in viral progeny occurs. An example of this latter type of virus is the phage lambda which on certain occasions, e.g., exposure to certain chemicals or radiation such as ultraviolet light, may initiate a lytic cycle immediately after infection but can otherwise exist as a provirus or prophage in the
E. coli
host genome for many generations.
A recent development in the field of antibodies is the amplification by the polymerase chain reaction (PCR) of nucleotide sequences for antibodies or portions thereof (1). An extension of this development is the insertion of these sequences into the genome of viruses, especially phages or bacteriophages (2, 3, 4, 11). In this regard reference is expressly made to PCT Patent Publication WO920 1047, published Jan. 23, 1992 entitled “Methods For Producing Members of Specific Binding Pairs,” incorporated herein by reference. Likewise, the expression of a catalytically-active enzyme on the surface of a phage has been achieved (32).
For instance, Clackson et al. (2) report using a random combinational library of rearranged sequences for heavy (V
h
) and kappa (V
k
) light chains from mice immune to the hapten 2-phenyloxazol-5-one (phOx) to display diverse libraries of antibody fragments on the surface of the fd phage. The recombinant fd phages were selected by passing the population thereof over an affinity column.
Likewise, McCafferty et al. (3) report that complete antibody V domains can be displayed on the surface of a recombinant fd bacteriophage and that those that bind to an antigen (e.g., one in a million) can be isolated by affinity chromatography. And, McCafferty et al. (32) report the expression and affinity chromatography of functional alkaline phosphatase on the surface of a bacteriophage.
Similarly, Huse et al. (4) relate employing the bacteriophage lambda vector system to express in
E. coli
a combinatorial library of Fab fragments. Selection for expression was by selection for binding to an antigen.
A problem with the technique of selection of recombinant phages suspected of expressing catalytic antibodies or catalytically active portions thereof by hapten or antigen binding or affinity is that initially an enormous number of phages are produced; for instance, of the order of greater than 10
5
. Selection for hapten-binding from this enormous population of phages still yields an enormous subpopulation of phages (that bind); for instance, of the order of 6,000-10,000 phages. However, in this first subpopulation that bind there is yet a smaller second subpopulation that not only express the antibody on their surface (and therefore bind to the hapten), but, also display a catalytic antibody (i.e., the antibody or portion thereof expressed is catalytic). Thus, isolation of only the first subpopulation (that bind with the antigen or hapten) does not adequately screen the recombinant phage population to isolate those members which express the antibody or portion thereof catalytically. That is, hapten-binding selection is insufficient to isolate those members of the recombinant phage population which express catalytic antibodies or portions thereof for further use; e.g., for infecting a host cell such as
E. coli
and producing consistent generations of recombinant phage or cells expressing the catalytic antibody or a catalytic portion thereof. Indeed, in a broader scope, a problem facing the development of catalysts such as catalytic antibodies, is the inability to economically enrich or select for moieties, e.g., antibodies, exhibiting the desired catalytic activity from among a vast excess of non-catalytic moieties, e.g., a vast excess of non-catalytic antibodies raised against the same transition state analogs.
Further, prior methods for selection of catalytic activity of antibody fragments (as opposed to their identification through extensive selection exercises) depends on biological selection based on the ability to compliment genetic defect in an organism expressing the fragment (16).
Heretofore there has been no method for selection of recombinant viruses or cells infected by such viruses displaying catalytic antibodies or catalytic portions thereof based upon catalytic properties of such viruses or cells.
In the area of enzymology the literature (5, 6) reports reactants called mechanism-based inhibitors (affinity labels or suicide substrates). These reactants bind in the active site of an enzyme as normal substrates do, but, contrary to normal substrates, exploit the chemical features of the reaction mechanism to form an irreversible adduct with the enzyme. Such reactants have been specifically designed for many enzymes and enzyme mechanisms. Generally, a nucleophilic enzyme amino acid residue that participates in the normal substrate catalytic reaction reacts instead with the mechanism-based inhibitor and is permanently inactivated. Haptens which were suicide substrates have been used to elicit antibodies (14). The suicide substrates were not used for selection of antibodies having catalytic activity.
Thus, heretofore there has been no application of mechanism-based inhibitors to select recombinant phage or recombinant phage infected cell populations for members expressing a catalytic antibody or catalytic portion thereof or to increase the concentration of members expressing catalytic moieties. Nor has there been any application of mechanism-based inhibitors to screen for catalytic moieties, such as catalytic moieties expressed by phages, cells, or other self-replicating systems, or catalytic peptides, oligopeptides, polypeptides, or enzymes. Nor has there been any application of mechanism-based inhibitors to increase the concentration of catalytic moieties in a sample containing catalytic moieties.
Work with enzymes show that active enzymes can “crawl” across a two-dimensional surface covered with substrate (on a micrometer distance scale), while inactive enzymes with the same binding affinity for the substrate
Blackburn Gary F.
Darsley Michael J.
Martin Mark T.
Simpson David M.
Smith Rodger G.
Evans, Esq. Barry
IGEN International Inc.
Kramer Levin Naftalis & Frankel LLP
Patterson Jr. Charles L.
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