GSJ:Received May 19, 2005: http://wbabin.net/saba/saba42.htm

Antibodies, and like molecules with highly specific binding to a target are valuable, for example in purifying a target protein. Previously a means of modulating the binding of an antibody was described (1).

Herein is detailed a method of isolating antibodies whose target binding is terminated via a sequence-specific protease.

Use of phage display to isolate protein sequences susceptible to a particular protease is a highly successful process (2,3). In this process, a phage library is constructed wherein each member has an amino-terminal domain which binds an affinity support, followed by a randomized peptide linker sequence, and then the carboxyl-terminal domain of M13 gene III. This phage library is contacted with a support-affixed target, and those phage bound are then treated with the protease. Those phage released often have linker peptide sequences susceptible to the protease.

In the present method, we start with a phage which expresses a single-strand antibody known to bind a particular target. The nucleic acid of this phage is then engineered such that a sequence-specific protease sequence is variously positioned within the amino acid sequence of the antibody to create a library. This library is then contacted with support-affixed target, and only those recombinants which still have the capacity to bind target do so. Finally, we subject the bound phage to the sequence-specific protease.

Figure 1 shows the process, wherein this particular target-bound phage is successfully released from the target.

Note this process need not involve proteases, but can also be used to find other agents which effect the release a target-bound protein. For example a physical characteristic such as heat, or a particular solvent or ligand.

Furthermore, notice a similar, but inverse process can be imagined for the isolation of antibodies which bind to target only in the presence of the modulating agent. Therein, one would first isolate those recombinant phage which could no longer bind target, and then select for target-binding phage with application of agent anticipated to cause the antibodies to bind target.

Notice that any target-binding protein, which is physically associated with it encoding nucleic acids, such as those produced in cis-display, could be utilized. Indeed, any means of encoding the target-binding recombinant proteins could be utilized to follow which have been modulated in their target binding.

Finally notice that molecules other than proteins could be the target-binder, for example nucleic acid aptamers.

This invention, as most inventions I've disclosed in this journal, is at the conceptual stage and a patent is anticipated. However, it is hoped that those with laboratories will investigate its full potential.

Provisional Claims

References.

1) Proteins Whose Functions are Modulated by Ligands or Enzymes.
Saba, JA Gen Sci J 2005 May 9

2) Substrate phage: selection of protease substrates by monovalent phage display.
Matthews, et al Science. 1993 May 21;260(5111):1113-7

3) Screening for protease substrate by polyvalent phage display.
Sedlacek, et al Comb Chem High Throughput Screen. 2005 Mar;8(2):197-203

4) Sequence specific Facter Xa Protease
(New England Biolabs)

5) Strategies for the construction and use of peptide and antibody libraries displayed on phages.
Pini, et al Curr Protein Pept Sci. 2004 Dec;5(6):487-96

6) Bivalent antibody phage display mimics natural immunoglobulin.
Lee, et al

Addendum 5/20/2005

Once an antibody with modulative affinity to a specific target has been found, several utilities other than target isolation are possible. For example, as depicted in Figure 2, the antibody could be conjugated to a material support or molecule such that the target is spatially confined till release from the antibody.

Exceptionally interesting possibilities, including diagnostic and therapeutic possibilities, arise if antibody binding to target also reversibly inactivates the functioning of the target.

Finally, one should make particular mention of yeast display of antibodies (1), and of course such a system could be utilized in the above method.

1) Yeast display of antibody fragments: a discovery and characterization platform.
Feldhaus, et al J Immunol Methods. 2004 Jul;290(1-2):69-80